US20040248796A1

US20040248796A1 - VEGF-B and PDGF modulation of stem cells

Info

Publication number: US20040248796A1
Application number: US10/772,927
Authority: US
Inventors: Kari Alitalo; Ulf Eriksson; Peter Carmeliet; Xuri Li; Desire Collen; Seppo Yla-Herttuala; Petri Salven; Iiro Rajantie
Original assignee: Vlaams Instituut voor Biotechnologie VIB; Ludwig Institute for Cancer Research Ltd; Licentia Oy
Current assignee: Vlaams Instituut voor Biotechnologie VIB; Ludwig Institute for Cancer Research Ltd; Licentia Oy
Priority date: 2003-02-04
Filing date: 2004-02-04
Publication date: 2004-12-09
Also published as: WO2004070018A3; JP2006517586A; WO2004070018A2; EP1594527A2; AU2004209668A1

Abstract

The present invention provides materials and methods for VEGF-B and PDGF therapy, especially therapy directed at stem cell recruitment, proliferation, and/or differentiation.

Description

BACKGROUND

This application claims the benefit of U.S. Provisional Application No. 60/445,021, filed Feb. 4, 2003, and U.S. Provisional Application No. 60/471,412, filed May 16, 2003, which are herein incorporated by reference in their entirety.

The platelet dervived growth factor (PDGF) proteins and their receptors (PDGFRs) are involved in regulation of cell proliferation, survival and migration of several cell types. The vascular endothelial growth factor (VEGF) proteins and their receptors (VEGFRs) play important roles in both vasculogenesis, the development of the embryonic vasculature from early differentiating endothelial cells, and angiogenesis, the process of forming new blood vessels from pre-existing ones [Risau, et al., Dev. Biol. 125:441-450 (1988); Zachary, Intl. J. Biochem. Cell. Bio. 30:1169-1174 (1998); Neufeld, et al., FASEB. J. 13:9-22 (1999); Ferrara, J. Mol. Med. 77:527-543 (1999)]. Both processes depend on the tightly controlled endothelial cell proliferation, migration, differentiation, and survival. Dysfunction of the endothelial cell regulatory system is a key feature of cancer and several diseases associated with abnormal angiogenesis, such as proliferative retinopathies, age-related macular degeneration, rheumatoid arthritis, and psoriasis. Understanding of the specific biological function of the key players involved in regulating endothelial cells will lead to more effective therapeutic applications to treat such diseases [Zachary, Intl. J. Biochem. Cell. Bio. 30:1169-1174 (1998); Neufeld et al., FASEB. J 13:9-22 (1999); Ferrara, J. Mol. Med. 77:527-543 (1999)].

Members of the PDGF/VEGF family are characterized by a number of structural motifs including a conserved PDGF motif defined by the sequence: P-[PS]-C—V-X(3)—R—C-[GSTA]-G-C—C, where the brackets indicate a variable position that can be any one of the amino acids within the brackets. The number contained within the parentheses indicates the number of amino acids that separate the “V” and “R” residues. This conserved motif falls within a large domain of 70-150 amino acids defined in part by eight highly conserved cysteine residues that form inter- and intramolecular disulfide bonds. This domain forms a cysteine knot motif composed of two disulfide bonds which form a covalently linked ring structure between two adjacent β strands, and a third disulfide bond that penetrates the ring [see for example, FIG. 1 in Muller et al., Structure 5:1325-1338 (1997)], similar to that found in other cysteine knot growth factors, e.g., transforming growth factor-β (TGF-β). The amino acid sequence of all known PDGF/VEGF proteins, with the exception of VEGF-E, contains the PDGF domain. The PDGF/VEGF family proteins are predominantly secreted glycoproteins that form either disulfide-linked or non-covalently bound homo- or heterodimers whose subunits are arranged in an anti-parallel manner [Stacker and Achen, Growth Factors 17:1-11 (1999); Muller et al., Structure 5:1325-1338 (1997)].

The platelet-derived growth factor (PDGF) subfamily comprises thus far four family members: PDGF-A, PDGF-B, PDGF-C, and PDGF-D. These ligands bind and activate, with distinct selectivity, dimeric complexes of the receptor tyrosine kinases PDGFR-α and PDGFR-β. [Heldin, C. H. & Westermark, B. Physiol Rev 79, 1283-1316 (1999).] PDGFR-A expression on cardiac vascular endothelial cells has been reported to be involved in the local communication among distinct cells in the heart [Edelberg, et al., J Clinical Inves. 102:837-43 (1998)]. The PDGFs regulate cell proliferation, cell survival and chemotaxis of many cell types in vitro (reviewed in [Heldin et al., Biochimica et Biophysica Acta 1378:F79-113 (1998); Carmeliet P et al. Nature 380, 435-9 (1996); Hellström, M. et al. J Cell Biol 153, 543-53. (2001).]. In vivo, the PDGF proteins exert their effects in a paracrine manner since they often are expressed in epithelial (PDGF-A) or endothelial (PDGF-B) cells in close apposition to the PDGF receptor-expressing mesenchyme [reviewed in Alitalo et al., Int Rev Cytology 172:95-127 (1997)]. Overexpression of the PDGFs has been observed in several pathological conditions, including malignancies, atherosclerosis, and fibroproliferative diseases. In tumor cells and cell lines grown in vitro, co-expression of the PDGFs and PDGF receptors generates autocrine loops, which are important for cellular transformation [Betsholtz et al., Cell 39:447-57 (1984); Keating et al., Science 239:914-6 (1988)]. PDGFR-α has a wide expression pattern [Heldin, C. H. & Westermark, B. Physiol. Rev. 79:1283-1316 (1999)].

The importance of the PDGFs as regulators of cell proliferation and cell survival is well illustrated by recent gene targeting studies in mice. Homozygous null mutations for either PDGF-A or PDGF-B are lethal in mice. Approximately 50% of the homozygous PDGF-A deficient mice have an early lethal phenotype, while the surviving animals have a complex postnatal phenotype with lung emphysema due to improper alveolar septum formation, and a dermal phenotype characterized by thin dermis, misshapen hair follicles, and thin hair. PDGF-A is also required for normal development of oligodendrocytes and subsequent myelination of the central nervous system. The PDGF-B deficient mice develop renal, hematological and cardiovascular abnormalities; where the renal and cardiovascular defects, at least in part, are due to the lack of proper recruitment of mural cells (vascular smooth muscle cells, pericytes or mesangial cells) to blood vessels.

PDGF-A and PDGF-B can homodimerize or heterodimerize to produce three different isoforms: PDGF-AA, PDGF-AB, or PDGF-BB. PDGF-A is only able to bind the PDGF α-receptor (PDGFR-α including PDGR-α/α homodimers). PDGF-B can bind both the PDGFR-α and a second PDGF receptor (PDGFR-β). More specifically, PDGF-B can bind to PDGFR-α/α and PDGFR-β/β homodimers, as well as PDGFR-α/β heterodimers. PDGF-C binds PDGR-α/α homodimers and PDGF-D binds PDGFR-β/β homodimers and both have been reported to bind PDGFR-α/β heterodimers.

PDGF-AA and -BB are the major mitogens and chemoattractants for cells of mesenchymal origin, but have no, or little effect on cells of endothelial lineage, although both PDGFR-α and -β are expressed on endothelial cells (EC). PDGF-BB and PDGF-AB have been shown to be involved in the stabilization/maturation of newly formed vessels [Isner, J. M. Nature 415, 234-9. (2002); Vale, P. R., Isner, J. M. & Rosenfield, K. J Interv Cardiol 14, 511-28 (2001); Heldin, C. H. & Westermark, B. Physiol Rev 79, 1283-1316 (1999); Betsholtz, C., Karlsson, L. & Lindahl, P. Bioessays 23, 494-507. (2001)]. Other data however, showed that PDGF-BB and PDGF-AA inhibited bFGF-induced angiogenesis in vivo via PDGFR-α signaling. PDGF-AA is among the most potent stimuli of mesenchymal cell migration, but it either does not stimulate or it minimally stimulates EC migration. In certain conditions, PDGF-AA even inhibits EC migration [Thommen, J Cell Biochem. 1997 Mar. 1;64(3):403-13; De Marchis, F., et al., Blood 99:2045-53 (2002); Cao, R., et al., FASEB. J 16:1575-83 (2002).] Moreover, PDGFR-α has been shown to antagonize the PDGFR-β-induced SMC migration Yu, J., et al., Biochem. Biophys. Res. Commun. 282:697-700 (2001) and neutralizing antibodies against PDGF-AA enhance smooth muscle cell (SMC) migration (Palumbo, R., et al., Arterioscler. Thromb. Vasc. Biol. 22:405-11 (2002). Thus, the angiogenic/arteriogenic activity of the PDGFs, especially when signaling through PDGFR-α, has been controversial and enigmatic.

PDGF-AA and -BB have been reported to play important roles in the proliferation and differentiation of both cardiovascular and neural stem/progenitor cells. PDGF-BB induced differentiation of Flk1+ embryonic stem cells into vascular mural cells [Carmeliet, P., Nature, 2000, 408:43-45; Yamashita, et al., Nature 408:92-6 (2000)], and potently increased neurosphere derived neuron survival [Caldwell, M. A. et al, Nat Biotechnol, 2001, 19:475-479]; while PDGF-AA stimulated oligodendrocyte precursor proliferation through α_vβ₃integrins [Baron, et al., Embo. J 21:1957-66 (2002)].

During development, PDGF-C is expressed in muscle progenitor cells and differentiated smooth muscle cells in most organs, including the heart, lung and kidney [Aase, K., et al., Mech. Dev. 110:187-91 (2002)]. In adulthood, PDGF-C is widely expressed in most organs, with the highest expression level in the heart and kidney [Li, X., et al., Nat. Cell. Biol. 2:302-09 (2000)]. PDGF-CC is secreted as an inactive homodimer of approximately 95 kD. Upon proteolytic removal of the CUB domain, PDGF-CC is capable of binding and activating its receptor, PDGFR-α [Li, X. & Eriksson, U., Cytokine & Growth Factor Reviews 244:1-8 (2003)]. In cells co-expressing both PDGFR-α and -β, PDGF-CC may also activate the PDGFR-α/β heterodimer, but not the PDGFR-β/β homodimer [Cao, R., et al., FASEB. J 16:1575-83. (2002); Gilbertson, D. G., et al., J. Biol. Chem. 10:10 (2001)].

Active PDGF-CC is a potent mitogen for fibroblast and vascular smooth muscle cells [Li, et al., Nat. Cell. Biol. 2:302-09 (2000); Cao, et al., FASEB. J 16:1575-83 (2002); Uutela, et al., Circulation 103:2242-7 (2001)]. Both PDGF-AA and PDGF-CC bind PDGFR-α, but only PDGF-CC potently stimulates angiogenesis in mouse cornea pocket and chick chorioallanoic membrane (CAM) assays [Cao, et al., FASEB. J 16:1575-83 (2002)]. PDGF-CC also promotes wound healing by stimulating tissue vascularization [Gilbertson, et al., J. Biol. Chem. 10:10 (2001)]. However, these studies did not address whether PDGF-CC stimulated vessel growth by affecting endothelial or smooth muscle cells, nor did they examine whether PDGF-CC promoted the maturation of newly formed vessels (including vasculogenesis, angiogenesis, neoangiogenesis and arteriogenesis).

The VEGF subfamily is composed of members that share a VEGF homology domain (VHD) characterized by the sequence: C—X(22-24)—P-[PSR]-C-V-X(3)—R—C-[GSTA]-G-C—C—X(6)—C—X(32-41)-C. The VHD domain, determined through analysis of the VEGF subfamily members, comprises the PDGF motif but is more specific. The VEGF subfamily of growth factors and receptors regulate the development and growth of the vascular endothelial system. VEGF family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF [Li, X. and U. Eriksson, “Novel VEGF Family Members: VEGF-B, VEGF-C and VEGF-D,” Int. J. Biochem. Cell. Biol., 33(4):421-6 (2001)).]

VEGF-A (or VEGF) was originally purified from several sources on the basis of its mitogenic activity toward endothelial cells, and also by its ability to induce microvascular permeability, hence it is also called vascular permeability factor (VPF). VEGF-A has subsequently been shown to induce a number of biological processes including the mobilization of intracellular calcium, the induction of plasminogen activator and plasminogen activator inhibitor-1 synthesis, promotion of monocyte migration in vitro, induction of antiapoptotic protein expression in human endothelial cells, induction of fenestrations in endothelial cells, promotion of cell adhesion molecule expression in endothelial cells and induction of nitric oxide mediated vasodilation and hypotension [Ferrara, J. Mol. Med. 77: 527-543 (1999); Neufeld, et al., FASEB. J 13:9-22 (1999); Zachary, Intl. J. Biochem. Cell. Bio. 30:1169-74 (1998)].

VEGF-A is a secreted, disulfide-linked homodimeric glycoprotein composed of 23 kD subunits. Five human VEGF-A isoforms of 121, 145, 165, 189 or 206 amino acids in length (VEGF121-VEGF206), encoded by distinct mRNA splice variants, have been described, all of which are capable of stimulating mitogenesis in endothelial cells. However, each isoform differs in biological activity, receptor specificity, and affinity for cell surface- and extracellular matrix-associated heparan-sulfate proteoglycans, which behave as low affinity receptors for VEGF-A. VEGF121 does not bind to either heparin or heparan-sulfate; VEGF145 and VEGF165 (GenBank Acc. No. M32977) are both capable of binding to heparin; and VEGF189 and VEGF206 show the strongest affinity for heparin and heparan-sulfates. VEGF121, VEGF145, and VEGF165 are secreted in a soluble form, although most of VEGF165 is confined to cell surface and extracellular matrix proteoglycans, whereas VEGF189 and VEGF206 remain associated with extracellular matrix. Both VEGF189 and VEGF206 can be released by treatment with heparin or heparinase, indicating that these isoforms are bound to extracellular matrix via proteoglycans. Cell-bound VEGF189 can also be cleaved by proteases such as plasmin, resulting in release of an active soluble VEGF110. Most tissues that express VEGF are observed to express several VEGF isoforms simultaneously, although VEGF121 and VEGF165 are the predominant forms, whereas VEGF206 is rarely detected [Ferrara, J. Mol. Med. 77:527-543 (1999)]. VEGF145 differs in that it is primarily expressed in cells derived from reproductive organs [Neufeld et al., FASEB. J 13:9-22 (1999)].

The pattern of VEGF-A expression suggests its involvement in the development and maintenance of the normal vascular system, and in angiogenesis associated with tumor growth and other pathological conditions such as rheumatoid arthritis. VEGF-A is expressed in embryonic tissues associated with the developing vascular system, and is secreted by numerous tumor cell lines. Analysis of mice in which VEGF-A was knocked out by targeted gene disruption indicate that VEGF-A is critical for survival, and that the development of the cardiovascular system is highly sensitive to VEGF-A concentration gradients. Mice lacking a single copy of VEGF-A die between day 11 and 12 of gestation. These embryos show impaired growth and several developmental abnormalities including defects in the developing cardiovasculature. VEGF-A is also required post-natally for growth, organ development, regulation of growth plate morphogenesis and endochondral bone formation. The requirement for VEGF-A decreases with age, especially after the fourth postnatal week. In mature animals, VEGF-A is required primarily for active angiogenesis in processes such as wound healing and the development of the corpus luteum. [Neufeld, et al., FASEB. J 13:9-22 (1999); Ferrara, J. Mol. Med. 77:527-543 (1999)]. VEGF-A expression is influenced primarily by hypoxia and a number of hormones and cytokines including epidermal growth factor (EGF), TGF-β, and various interleukins. Regulation occurs transcriptionally and also post-transcriptionally such as by increased mRNA stability [Ferrara, J. Mol. Med. 77:527-543 (1999)].

Lack of a single VEGF (VEGF-A) allele results in embryonic lethality (Canneliet, P., et al., Nature, 380(6573):435-39 (1996); and Ferrara, N., et al., Nature, 380(6573):439-42 (1996)). VEGF-A binds to four receptors, VEGFR-1, VEGFR-2, neuropilin-1 and neuropilin-2 (Poltorak, Z., T. Cohen, and G. Neufeld, Herz., 25(2):126-9 (2000)).

PlGF, another member of the VEGF subfamily, is generally a poor stimulator of angiogenesis and endothelial cell proliferation in comparison to VEGF-A, and the in vivo role of PlGF is not well understood. Three isoforms of PlGF produced by alternative mRNA splicing have been described [Hauser, et al., Growth Factors 9:259-268 (1993); Maglione, et al., Oncogene 8:925-931 (1993)]. PlGF forms both disulfide-liked homodimers and heterodimers with VEGF-A. The PlGF-VEGF-A heterodimers are more effective at inducing endothelial cell proliferation and angiogenesis than PlGF homodimers. PlGF is primarily expressed in the placenta, and is also co-expressed with VEGF-A during early embryogenesis in the trophoblastic giant cells of the parietal yolk sac [Stacker and Achen, Growth Factors 17:1-11 (1999)].

For sometime, research on the control of vessel growth focused on VEGF and VEGFR-2, but recently more attention has been given to VEGFR-1 and its ligands besides VEGF, including PlGF and VEGF-B. [Eriksson and Alitalo, Nat. Med. 8:775-777 (2002).] PlGF knock out mice do not experience significant abnormalities in embryonic angiogenesis. However, PlGF deficiency in mice has been reported to impair angiogenesis, plasma extravasation and collateral growth during ischemia, inflammation, wound healing and cancer. [Cammeliet, et al., Nat. Med. 7:575-83 (2001).] Hattori, et al. have reported that PlGF promotes the recruitment of VEGFR-1+hematopoietic stem cells from a quiescent to a proliferative bone marrow microenvironment, contributing to hematopoiesis. [Nat. Med. 8:841-49 (2002).] Luttun and co-workers have reported that PlGF stimulated angiogenesis and collateral growth in ischemic heart and limb with an efficiency comparable to, if not higher than, that of VEGF. [Nat. Med. 8:831-40 (2002).]

The isolation and characteristics of VEGF-B, including nucleotide and amino acid sequences for both human and murine VEGF-B, are described in detail in PCT/US96/02957, and U.S. Pat. Nos. 5,840,693 and 5,607,918 by Ludwig Institute for Cancer Research and Helsinki University Licensing Ltd. Oy and in Olofsson, et al., Proc. Natl. Acad. Sci. USA, 93:2576-2581 (1996). A nucleotide sequence encoding human VEGF-B is also found at GenBank Accession No. U48801. The entire disclosures of the International Patent Application PCT/US97/14696 (WO 98/07832), U.S. Pat. Nos. 5,840,693 and 5,607,918 are incorporated herein by reference.

VEGF-B is very strongly expressed in the heart, and only weakly in the lungs, whereas the reverse is the case for VEGF-A. RT-PCR assays have demonstrated the presence of VEGF-B mRNA in melanoma, normal skin, and muscle. This suggests that VEGF-A and VEGF-B, despite the fact that they are co-expressed in many tissues, have functional differences. A comparison of the PDGF/VEGF family of growth factors reveals that the 167 amino acid isoform of VEGF-B is the only family member that is completely devoid of any glycosylation. Gene targeting studies have shown that VEGF-B deficiency results in mild cardiac phenotype, and impaired coronary vasculature (Bellomo, et al., Circ. Res., 86:E29-35 (2000)).

The human and murine genes for VEGF-B are almost identical, and both span about 4 kb of DNA. The genes are composed of seven exons, and their exon-intron organization resembles that of the VEGF-A and PlGF genes. [Grimmond, et al., Genome Res., 6:124-131 (1996); Olofsson, et al., J. Biol. Chem., 271:19310-17 1996); Townson, et al., Biochem. Biophys. Res. Commun. 220:922-928 (1996).]VEGF-B binds specifically to VEGFR-1 and neuropilin-1. [Olofsson, B., et al., Proc. Nat'l. Acad. Sci. USA, 93(6):2576-81 (1996); Olofsson, B., et al., Proc. Nat'l. Acad. Sci. USA, 95(20):11709-14 (1998).]

VEGF-B displays a unique expression pattern compared with other VEGF family members, with the highest expression level in the cardiac myocytes [Aase, K., et al., Developmental Dynamics, 215(1):12-25 (1999)], whereas VEGFR-1 is expressed in the adjacent endothelial cells [Aase, K., et al., Developmental Dynamics, 215(1):12-25 (1999)], and neuropilin-1 (NP-1) is expressed in both endothelium and cardiac myocytes during development. [Makinen, T., et al., Journal of Biological Chemistry, 274(30):21217-22 (1999); and Kitsukawa, T., et al., Development, 121(12):4309-18 (1995).] The temporal-spatial expression patterns of VEGF-B and its receptors suggest both autocrine and paracrine roles of VEGF-B in the heart.

Both VEGF-B and PlGF exist in two alternatively spliced forms, which differ in their affinity for heparin, and both growth factors are able to form heterodimers with VEGF. Olofsson, et al., Cell Biol., 95:11709-11714 (1998). Although VEGF-B and PlGF both appear to bind exclusively to VEGFR-1 and not VEGFR-2 or VEGFR-3, the two growth factors appear to have different functions. For example, Hattori et al., have reported that PlGF affects hematopoiesis recovery by both binding to VEGFR-1 and by inducing expression of matrix metalloproteinase-9. [Nat. Med. 8:841-49 (2002).] Carmeliet, et al., reported that VEGF-B did not rescue development in PlGF deficient mice. [Nat. Med., 7:575-83 (2001).] The expression of VEGF-B and PlGF are also substantially different with VEGF-B, unlike PlGF, widely expressed and most prominently in heart and skeletal muscle. Furthermore, VEGF residues implicated in VEGFR-1 binding are more highly conserved in VEGF-B than in PlGF. [Olofsson, et al., Cell Biol. 95:11709-11714 (1998).] Proteolytic processing is required for VEGF-B₁₈₆, a VEGF-β isoform discussed below, to bind NP-1, but no such processing is required for PlGF to bind NP-1.

Two VEGF-β isoforms generated by alternative mRNA splicing exist, VEGF-B ₁₈₆and VEGF-B₁₆₇, with the first isoform accounting for about 80% of the total VEGF-B transcripts [Li, X., et al., Growth Factor, 19:49-59 (2001).] [Grimmond, et al., Genome Res., 6:124-131 (1996); Olofsson, et al., J. Biol. Chem., 271:19310-19317 (1996).] The isoforms have an identical N-terminal domain of 115 amino acid residues, excluding the signal sequence. The common N-terminal domain is encoded by exons 1-5. Differential use of the remaining exons 6A, 6B and 7 gives rise to the two splice isoforms. By the use of an alternative splice-acceptor site in exon 6, an insertion of 101 bp introduces a frame-shift and a stop of the coding region of VEGF-B₁₆₇cDNA. Thus, the two VEGF-β isoforms have differing C-terminal domains.

The two VEGF-β isoforms differ at their carboxy-termini and display different abilities to bind neuropilin-1. [Makinen, et al., J. Biol. Chem., 274(30):21217-22 (1999).] Moreover, VEGF-B₁₈₆is freely secreted, while VEGF-B₁₆₇is secreted but largely cell-associated, implying that the functional properties of the two isoforms may be distinct. Both isoforms bind to extracellular matrix tenascin-X and stimulate endothelial cell proliferation through VEGF-receptor-1 (VEGFR-1). [Ikuta, et al., Genes Cells, 5(11):913-927 (2000).]

The different C-terminal domains of the two splice isoforms of VEGF-B affect their biochemical and cell biological properties. The C-terminal domain of VEGF-B ₁₆₇is structurally related to the corresponding region in VEGF, with several conserved cysteine residues and stretches of basic amino acid residues. Thus, this domain is highly hydrophilic and basic and, accordingly, VEGF-B₁₆₇will remain cell-associated on secretion, unless the producing cells are treated with heparin or high salt concentrations. The cell-associated molecules binding VEGF-B₁₆₇are likely to be cell surface or pericellular heparin sulfate proteoglycans. It is likely that the cell-association of this isoform occurs via its unique basic C-terminal region. The hydrophobic C-terminal domain of VEGF-B₁₈₆has no significant similarity with known amino acid sequences in the databases. VEGF-B₁₈₆is freely secreted from cells [(Olfsson et al., J. Biol. Chem.,271:19310-19317 (1996)] and evidence indicates that this isoform is proteolytically processed, regulating the biological properties of the protein. [Olofsson, et al., Proc. Natl. Acad. Sci. USA, 95:11709-11714 (1998).]

A further difference between the VEGF-β isoforms is found in the glycosylation of the VEGF-β isoforms. VEGF-B ₁₆₇is not glycosylated at all, whereas VEGF-B₁₈₆is O-glycosylated but not N-glycosylated.

Both isoforms of VEGF-B can form heterodimers with VEGF, consistent with the conservation of the eight cysteine residues involved in inter- and intramolecular disulfide bonding of PDGF-like proteins. Furthermore, co-expression of VEGF-B and VEGF in many tissues suggests that VEGF-B-VEGF heterodimers occur naturally. Heterodimers of VEGF-B ₁₆₇-VEGF remain cell-associated. In contrast, heterodimers of VEGF-B₁₈₆and VEGF are freely secreted from cells in a culture medium. VEGF also forms heterodimers with PlGF. [DiSalvo, et al, J. Biol. Chem. 270:7717-7723 (1995).] The production of heterodimeric complexes between the members of this family of growth factors could provide a basis for a diverse array of angiogenic or regulatory molecules. Enholm, et al., WO 02/36131 report adenovirus gene therapy using a first vector encoding VEGF-B together with a second vector encoding another vascular endothelial growth factor to stimulate angiogenic activity.

A fourth member of the VEGF subfamily, VEGF-C, comprises a VHD that is approximately 30% identical at the amino acid level to VEGF-A. VEGF-C is originally expressed as a larger precursor protein, prepro-VEGF-C, having extensive amino- and carboxy-terminal peptide sequences flanking the VHD, with the C-terminal peptide containing tandemly repeated cysteine residues in a motif typical of Balbiani ring 3 protein. Prepro-VEGF-C undergoes extensive proteolytic maturation involving the successive cleavage of a signal peptide, the C-terminal pro-peptide, and the N-terminal pro-peptide. Secreted VEGF-C protein consists of a non-covalently-linked homodimer, in which each monomer contains the VHD. The intermediate forms of VEGF-C produced by partial proteolytic processing show increasing affinity for the VEGFR-3 receptor, and the mature protein is also able to bind to the VEGFR-2 receptor. [Joukov, et al., EMBO J, 16(13):3898-3911 (1997).] It has also been demonstrated that a mutant VEGF-C, in which a single cysteine at position 156 is either substituted by another amino acid or deleted, loses the ability to bind VEGFR-2 but remains capable of binding and activating VEGFR-3 [International Patent Publication No. WO 98/33917]. In mouse embryos, VEGF-C mRNA is expressed primarily in the allantois, jugular area, and the metanephros. [Joukov, et al., J. Cell. Physiol. 173:211-15 (1997)].

VEGF-C is involved in the regulation of lymphatic angiogenesis: when VEGF-C was overexpressed in the skin of transgenic mice, a hyperplastic lymphatic vessel network was observed, suggesting that VEGF-C induces lymphatic growth [Jeltsch et al., Science, 276:1423-1425 (1997)]. Continued expression of VEGF-C in the adult also indicates a role in maintenance of differentiated lymphatic endothelium [Ferrara, J. Mol. Med. 77:527-543 (1999)]. In addition, VEGF-C shows angiogenic properties: it can stimulate migration of bovine capillary endothelial (BCE) cells in collagen and promote growth of human endothelial cells. [See, e.g., International Patent Publication No. WO 98/33917, incorporated herein by reference.]

VEGF-D is structurally and functionally most closely related to VEGF-C. [See International Patent Publ. No. WO 98/07832, incorporated herein by reference]. Like VEGF-C, VEGF-D is initially expressed as a prepro-peptide that undergoes N-terminal and C-terminal proteolytic processing, and forms non-covalently linked dimers. VEGF-D stimulates mitogenic responses in endothelial cells in vitro. During embryogenesis, VEGF-D is expressed in a complex temporal and spatial pattern, and its expression persists in the heart, lung, and skeletal muscles in adults. Isolation of a biologically active fragment of VEGF-D designated VEGF-DΔNΔC, is described in International Patent Publication No. WO 98/07832, incorporated herein by reference. VEGF-DANAC consists of amino acid residues 93 to 201 of VEGF-D linked to the affinity tag peptide FLAG®.

Four additional members of the VEGF subfamily have been identified in poxviruses, which infect humans, sheep and goats. The orf virus-encoded VEGF-E and NZ2 VEGF are potent mitogens and permeability enhancing factors. Both show approximately 25% amino acid identity to mammalian VEGF-A, and are expressed as disulfide-liked homodimers. Infection by these viruses is characterized by pustular dermatitis which may involve endothelial cell proliferation and vascular permeability induced by these viral VEGF proteins. [Ferrara, J. Mol. Med. 77:527-543 (1999); Stacker and Achen, Growth Factors 17:1-11 (1999)]. VEGF-like proteins have also been identified from two additional strains of the orf virus, D1701 [GenBank Acc. No. AF106020; described in Meyer, et al., EMBO. J 18:363-374 (1999)] and NZ10 [described in International Patent Application PCT/US99/25869, incorporated herein by reference]. These viral VEGF-like proteins have been shown to bind VEGFR-2 present on host endothelium, and this binding is important for development of infection and viral induction of angiogenesis. [Meyer, et al., EMBO. J 18:363-74 (1999); International Patent Application PCT/US99/25869.]

Seven cell surface receptors that interact with PDGF/VEGF family members have been identified. These include PDGFR-α [see e.g., GenBank Acc. No. NM006206], PDGFR-β [see e.g., GenBank Acc. No. NM002609], VEGFR-1/Flt-1 (fms-like tyrosine kinase-1) [GenBank Acc. No. X51602; De Vries, et al., Science 255:989-991 (1992)]; VEGFR-2/KDR/Flk-1 (kinase insert domain containing receptor/fetal liver kinase-1) [GenBank Acc. Nos. X59397 (Flk-1) and L04947 (KDR); Terman, et al., Biochem. Biophys. Res. Comm. 187:1579-1586 (1992); Matthews, et al., Proc. Natl. Acad. Sci. USA 88:9026-9030 (1991)]; VEGFR-3/Flt4 (fms-like tyrosine kinase 4) [U.S. Pat. No. 5,776,755 and GenBank Acc. No. X68203 and S66407; Pajusola et al., Oncogene 9:3545-3555 (1994)]; neuropilin-1 [Gen Bank Acc. No. NM003873], and neuropilin-2 [Gen Bank Acc. No. NM003872]. The two PDGF receptors mediate signaling of PDGFs as described herein. VEGF121, VEGF165, VEGF-B, PlGF-1 and PlGF-2 bind VEGF-R1; VEGF121, VEGF145, VEGF165, VEGF-C, VEGF-D, VEGF-E, and NZ2 VEGF bind VEGF-R2; VEGF-C and VEGF-D bind VEGFR-3; VEGF165, PlGF-2, and NZ2 VEGF bind neuropilin-1; and VEGF165 binds neuropilin-2.[Neufeld, et al., FASEB. J 13:9-22 (1999); Stacker and Achen, Growth Factors 17:1-11 (1999); Ortega, et al., Fron. Biosci. 4:141-152 (1999); Zachary, Intl. J. Biochem. Cell. Bio. 30:1169-1174 (1998); Petrova, et al., Exp. Cell. Res. 253:117-130 (1999)].

The PDGF receptors (including PDGFR-α/α, PDGFR-α/β, and PDGFR-β/β) are protein tyrosine kinase receptors (PTKS) that contain five immunoglobulin-like loops in each of their extracellular domains. VEGFR-1, VEGFR-2, and VEGFR-3 comprise PTKs that are distinguished by the presence of seven Ig domains in their extracellular domain and a split kinase domain in the cytoplasmic region. Both neuropilin-1 and neuropilin-2 are non-PTK VEGF receptors. NP-1 has an extracellular portion includes a MAM domain; regions of homology to coagulation factors V and VIII, MFGPs and the DDR tyrosine kinase; and two CUB-like domains.

Several of the VEGF receptors are expressed as more than one isoform. A soluble isoform of VEGFR-1 lacking the seventh Ig-like loop, transmembrane domain, and the cytoplasmic region is expressed in human umbilical vein endothelial cells. This VEGFR-1 isoform binds VEGF-A with high affinity and is capable of preventing VEGF-A-induced mitogenic responses [Ferrara, J. Mol. Med. 77:527-543 (1999); Zachary, Intl. J. Biochem. Cell. Bio. 30:1169-1174 (1998)]. A C-terminal truncated from of VEGFR-2 has also been reported [Zachary, Intl. J. Biochem. Cell. Bio. 30:1169-1174 (1998)]. In humans, there are two isoforms of the VEGFR-3 protein which differ in the length of their C-terminal ends. Studies suggest that the longer isoform is responsible for most of the biological properties of VEGFR-3.

The receptors for the PDGFs, PDGF α-receptor (PDGFR-α) and the α-receptor (PDGFR-β), are expressed by many in vitro grown cell lines, and they are mainly expressed by mesenchymal cells in vivo.

Gene targeting studies in mice have revealed distinct physiological roles for the PDGF receptors despite the overlapping ligand specificities of the PDGFRs [Rosenkranz, et al., Growth Factors 16:201-16 (1999)]. Homozygous null mutations for either of the two PDGF receptors are lethal. PDGFR-β deficient mice die during embryogenesis at day 10, and show incomplete cephalic closure, impaired neural crest development, cardiovascular defects, skeletal defects, and edemas. The PDGFR-β deficient mice develop similar phenotypes to animals deficient in PDGF-B, that are characterized by renal, hematological and cardiovascular abnormalities; where the renal and cardiovascular defects, at least in part, are due to the lack of proper recruitment of mural cells (vascular smooth muscle cells, pericytes or mesangial cells) to blood vessels.

The expression of VEGFR-1 occurs mainly in vascular endothelial cells, although some may be present on monocytes, trophoblast cells, and renal mesangial cells [Neufeld et al., FASEB. J 13:9-22 (1999)]. High levels of VEGFR-1 mRNA are also detected in adult organs, suggesting that VEGFR-1 has a function in quiescent endothelium of mature vessels not related to cell growth. VEGFR-1−/− mice die in utero between day 8.5 and 9.5. Although endothelial cells developed in these animals, the formation of functional blood vessels was severely impaired, suggesting that VEGFR-1 may be involved in cell-cell or cell-matrix interactions associated with cell migration. Recently, it has been demonstrated that mice expressing a mutated VEGFR-1 in which only the tyrosine kinase domain was missing show normal angiogenesis and survival, suggesting that the signaling capability of VEGFR-1 is not essential. [Neufeld, et al., FASEB. J 13:9-22 (1999); Ferrara, J. Mol. Med. 77:527-543 (1999)].

VEGFR-2 expression is similar to that of VEGFR-1 in that it is broadly expressed in the vascular endothelium, but it is also present in hematopoietic stem cells, megakaryocytes, and retinal progenitor cells [Neufeld, et al., FASEB. J 13:9-22 (1999)]. Although the expression pattern of VEGFR-1 and VEGFR-2 overlap extensively, evidence suggests that, in most cell types, VEGFR-2 is the major receptor through which most of the VEGFs exert their biological activities. Examination of mouse embryos deficient in VEGFR-2 further indicate that this receptor is required for both endothelial cell differentiation and the development of hematopoietic cells [Joukov, et al., J. Cell. Physiol. 173:211-215 (1997)].

VEGFR-3 is expressed broadly in endothelial cells during early embryogenesis. During later stages of development, the expression of VEGFR-3 becomes restricted to developing lymphatic vessels [Kaipainen, A., et al., Proc. Natl. Acad. Sci. USA 92:3566-70 (1995)]. In adults, the lymphatic endothelia and some high endothelial venules express VEGFR-3, and increased expression occurs in lymphatic sinuses in metastatic lymph nodes and in lymphangioma. VEGFR-3 is also expressed in a subset of CD34+hematopoietic cells which may mediate the myelopoietic activity of VEGF-C demonstrated by overexpression studies [WO 98/33917]. Targeted disruption of the VEGFR-3 gene in mouse embryos leads to failure of the remodeling of the primary vascular network, and death after embryonic day 9.5 [Dumont, et al., Science 282:946-49 (1998)]. These studies suggest an essential role for VEGFR-3 in the development of the embryonic vasculature, and also during lymphangiogenesis.

Structural analyses of the VEGF receptors indicate that the VEGF-A binding site on VEGFR-1 and VEGFR-2 is located in the second and third Ig-like loops. Similarly, the VEGF-C and VEGF-D binding sites on VEGFR-2 and VEGFR-3 are also contained within the second Ig-loop [Taipale, et al., Curr. Top. Microbiol. Immunol. 237:85-96 (1999)]. The second Ig-like loop also confers ligand specificity as shown by domain swapping experiments [Ferrara, J. Mol. Med. 77:527-543 (1999)]. Receptor-ligand studies indicate that dimers formed by the VEGF family proteins are capable of binding two VEGF receptor molecules, thereby dimerizing VEGF receptors. The fourth Ig-like loop on VEGFR-1, and also possibly on VEGFR-2, acts as the receptor dimerization domain that links two receptor molecules upon binding of the receptors to a ligand dimer [Ferrara, J. Mol. Med. 77:527-543 (1999)]. Although the regions of VEGF-A that bind VEGFR-1 and VEGFR-2 overlap to a large extent, studies have revealed two separate domains within VEGF-A that interact with either VEGFR-1 or VEGFR-2, as well as specific amino acid residues within these domains that are critical for ligand-receptor interactions. Mutations within either VEGF receptor-specific domain that specifically prevent binding to one particular VEGF receptor have also been recovered [Neufeld, et al., FASEB. J 13:9-22 (1999)].

VEGFR-1 and VEGFR-2 are structurally similar, share common ligands (VEGF121 and VEGF165), and exhibit similar expression patterns during development. However, the signals mediated through VEGFR-1 and VEGFR-2 by the same ligand appear to be slightly different. VEGFR-2 has been shown to undergo autophosphorylation in response to VEGF-A, but phosphorylation of VEGFR-1 under identical conditions was barely detectable. VEGFR-2 mediated signals cause striking changes in the morphology, actin reorganization, and membrane ruffling of porcine aortic endothelial cells recombinantly overexpressing this receptor. In these cells, VEGFR-2 also mediated ligand-induced chemotaxis and mitogenicity; whereas VEGFR-1-transfected cells lacked mitogenic responses to VEGF-A. Mutations in VEGF-A that disrupt binding to VEGFR-2 fail to induce proliferation of endothelial cells, whereas VEGF-A mutants that are deficient in binding VEGFR-1 are still capable of promoting endothelial proliferation. Similarly, VEGF stimulation of cells expressing only VEGFR-2 leads to a mitogenic response whereas comparable stimulation of cells expressing only VEGFR-1 also results in cell migration, but does not induce cell proliferation. In addition, phosphoproteins co-precipitating with VEGFR-1 and VEGFR-2 are distinct, suggesting that different signaling molecules interact with receptor-specific intracellular sequences.

The primary function of VEGFR-1 in angiogenesis may be to negatively regulate the activity of VEGF-A by binding it and thus preventing its interaction with VEGFR-2, whereas VEGFR-2 is thought to be the main transducer of VEGF-A signals in endothelial cells. In support of this hypothesis, mice deficient in VEGFR-1 die as embryos while mice expressing a VEGFR-1 receptor capable of binding VEGF-A but lacking the tyrosine kinase domain survive and do not exhibit abnormal embryonic development or angiogenesis. In addition, analyses of VEGF-A mutants that bind only VEGFR-2 show that they retain the ability to induce mitogenic responses in endothelial cells. However, VEGF-mediated migration of monocytes is dependent on VEGFR-1, indicating that signaling through this receptor is important for at least one biological function. In addition, the ability of VEGF-A to prevent the maturation of dendritic cells is also associated with VEGFR-1 signaling, suggesting that VEGFR-1 may function in cell types other than endothelial cells. [Ferrara, J. Mol. Med. 77:527-543 (1999); Zachary, Intl. J. Biochem. Cell. Bio. 30:1169-1174 (1998)].

Neuropilin-1 was originally cloned as a receptor for the collapsin/semaphorin family of proteins involved in axon guidance [Stacker and Achen, Growth Factors 17:1-11 (1999)]. It is expressed in both endothelia and specific subsets of neurons during embryogenesis, and it thought to be involved in coordinating the developing neuronal and vascular system. Although activation of neuropilin-1 does not appear to elicit biological responses in the absence of the VEGF family tyrosine-kinase receptors, their presence on cells leads to more efficient binding of VEGF165 and VEGFR-2 mediated responses. [Neufeld, et al., FASEB. J. 13:9-22 (1999)] Mice lacking neuropilin-1 show abnormalities in the developing embryonic cardiovascular system. [Neufeld, et al., FASEB. J. 13:9-22 (1999)] Neuropilin-2 was identified by expression cloning and is a collapsin/semaphorin receptor closely related to neuropilin-1. Neuropilin-2 is an isoform-specific VEGF receptor in that it only binds VEGF165. Like neuropilin-1, neuropilin-2 is expressed in both endothelia and specific neurons, and is not predicted to function independently due to its relatively short intracellular domain. The function of neuropilin-2 in vascular development is unknown [Neufeld, et al., FASEB. J. 13:9-22 (1999); WO 99/30157].

Stem cells, also referred to as progenitor cells, comprise both embryonic and adult stem cells. Adult stems cells include, but are not limited to, neural stem cells, hematopoietic stem cells, endothelial stem cells, and epithelial stem cells. See Tepper, et al., Plastic and Reconstructive Surgery, 111:846-854 (2003). Endothelial progenitor cells circulate in the blood and migrate to regions characterized by injured endothelia. Kaushal, et al., Nat. Med., 7:1035-1040 (2001). A small subpopulation of human CD34(+)CD133(+) stem cells from different hematopoietic sources coexpress VEGFR-3 (Salven, et al., Blood, 101(1):168-72 (2003). These cells also have the capacity to differentiate to lymphatic and/or vascular endothelial cells in vitro.

Myelosuppression or bone marrow suppression is a problem experienced by those subjects undergoing chemotherapy and bone marrow transplants. New methods of treating myelosuppression are needed in the art.

There remains a need in the art for new therapies employing VEGF-B and PDGFs or antagonists of VEGF-B and PDGFs. There is also a need in the art to identify growth factors capable of causing mobilization of vascular stem/progenitor cells, increase of stem cell adherence/viability, and promotion of stem cell differentiation for use in therapies.

For therapeutic revascularization of ischemic tissues to succeed, the newly formed vessels must be mature, durable and functional. These requirements imply not only that new endothelium-lined vessels must sprout (“angiogenesis”), but also that these nascent vessels become covered by perivascular smooth muscle cells and/or fibroblasts (“arteriogenesis”), processes that require an involvement of both vascular progenitors and differentiated cells of multiple cardiovascular cell types. Neoangiogenesis and vasculogenesis are also relevant. Vasculogenesis, including adult vasculogenesis, is a process by vascular progenitors are differentiated and mobilized to sites of active vessel growth. While angio/arteriogenesis are easily disregulated by inactivation of candidate genes [Carmeliet, P., et al. Nature 380:435-39 (1996); Hellström, M., et al., J. Cell. Biol. 153:543-53 (2001)], stimulating these processes in a functionally relevant manner has proven to be a much greater challenge than anticipated. A need exists for materials and methods for meeting this goal, preferentially those having pleiotropic activities on both vascular progenitors and differentiated vascular cells of both endothelial and smooth muscle cell lineages.

Neither VEGF, PDGF-AA, PDGF-BB, TGF-β, bFGF nor PlGF has been documented to induce the expression of SMC genes in adult bone marrow-derived progenitors, and very few molecules have been discovered to regulate the differentiation and function of SMC progenitors derived from adult bone marrow (BM) stem cells. [Hirschi, K. K. & Goodell, M. A., Gene Ther. 9:648-52 (2002).] PDGF-BB stimulates embryonic vascular progenitors to acquire a SMC-phenotype [Hirschi, K. K. & Goodell, M. A., Gene Ther. 9:648-52 (2002); Carmeliet, P., Nature 408:43, 45 (2000); Yamashita, J., et al. Nature 408:92-6 (2000)], but is unknown to have similar effects on adult bone marrow-derived progenitors. Thus, there is a need to identify and characterize molecules capable of affecting adult bone marrow-derived progenitors, for use in diagnosis, medicament preparation, and therapy.

SUMMARY OF THE INVENTION

The present invention relates to new methods of modulating progenitor cell recruitment, proliferation, and/or differentiation, and is based in part on the discovery that VEGF-B and PDGF-C stimulate the recruitment, proliferation and/or differentiation of stem cells. Each of these growth factors may be used alone or in combination with other growth factors as described herein.

In one aspect of the invention VEGF-B is used to stimulate the recruitment, proliferation and/or differentiation of stem cells, including hematopoietic and endothelial precursor cells. In one embodiment, a method of stimulating stem cell recruitment, proliferation, or differentiation is provided which comprises identifying a human subject in need of stem cell recruitment, proliferation, or differentiation, and administering to the human subject a composition comprising a vascular endothelial growth factor B (VEGF-B) product. The term “stem cell recruitment” refers to mobilization of stem cells (e.g., from bone marrow into circulation). The term “proliferation” refers to mitotic reproduction. The term “differentiation” refers to the process by which the pluripotent or multipotent stem cells develop into other cell types. Differentiation may involve a number of stages between pluripotency and fully differentiated cell types, and stimulation through even one stage is considered stimulating differentiation. The terms “proliferation” and “differentiation” are relevant in both in vivo and ex vivo therapies. The recruitment, proliferation, and differentiation are all relevant to the process of myelopoiesis—involving the formation and development of white blood cells.

The identifying step involves a medical diagnosis to identify a subject that suffers from a disease or condition that would benefit from stem cell recruitment, proliferation, or differentiation. For example, it is known that myelosuppression, which is characterized by reduced white blood cell counts and may be due to reduced production of such cells from stem cells or bone marrow origin, is a serious side effect of many cancer chemotherapy drugs. Thus, in one variation, the identifying step comprises selecting a human subject undergoing antineoplastic chemotherapy. The administering of the VEGF-B product to such a subject can be performed before, during, or after a chemotherapy dosing. VEGF-B product administration contemporaneously with, or after, administering the antineoplastic chemotherapy is preferred. The VEGF-B product is preferably administered in a dosing regimen to promote myelopoiesis.

Re-establishment of a healthy white blood cell count is an important clinical consideration for patients undergoing radiation therapy. Thus, in another variation, the identifying comprises selecting a subject undergoing radiation thereapy as the candidate for VEGF-B therapy. The VEGF-B product is preferably administered contemporaneously with or after the radiation therapy.

Similarly, re-establishment of a healthy white blood cell count is critical for bone marrow transplant patents. Thus, in another variation, the identifying comprises selecting a bone marrow transplant subject as the candidate for VEGF-B therapy. The VEGF-B product is preferably administered contemporaneously with or after the bone marrow transplant. Other patient populations include individuals that are immunosuppressed for any reason, e.g., due to infection with a human immunodeficiency virus (HIV, AIDS).

Vascular endothelial growth factor B (VEGF-B) is a naturally-occurring protein in humans and other animals, encoded by a gene in the human/animal genome. VEGF-B binds to receptor VEGFR-1 and is described in greater detail below. The term “VEGF-B product” encompasses both VEGF-B polypeptide materials as described in greater detail below, and polynucleotides that encode VEGF-B polypeptides.

Thus, in one variation, the VEGF-B product comprises a VEGF-B polypeptide. For the purposes of the invention, VEGF-B (also known as VEGF-related factor (VRF)) refers to proteins having the same amino acid sequence as a naturally-occurring VEGF-B protein, and also fragments, analogs, or variants that have sequence variation, yet retain VEGFR-1 binding affinity. In one variation, the VEGF polypeptide is glycosylated. Exemplary glycosylated VEGF-B forms are described in published U.S. Patent Application No. 2002/0068694 and U.S. Patent. Nos. 5,607,918, 5,840,693, and 5,928,939, all incorporated by reference. In a preferred embodiment, the VEGF-B polypeptide has an amino acid sequence at least 85% or 90% identical to a natural human VEGF-B sequence. Still more preferred are those polypeptides that are 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical at the amino acid sequence level with a naturally-occurring human VEGF-B sequence. Exemplary human VEGF-β isoforms comprise the sequences set forth in SEQ ID NOS: 2 and 4, wherein secreted mature forms begin with amino acid position 1. Nucleotide and deduced amino acid sequences for VEGF-B are deposited in GenBank under Acc. No. U48801.

The sequence variation that is contemplated also can be defined in terms of the polynucleotide that encodes the VEGF-B polypeptide. For example, the VEGF-B polypeptide binds VEGFR-1 and is preferably encoded by a polynucleotide that hybridizes under stringent conditions with the complement of the polynucleotide in SEQ ID NO: 1 or 3, both of which correspond to human VEGF-B sequences. Exemplary stringent conditions are provided below.

In another variation of the invention, the VEGF-B product comprises a polynucleotide that encodes a VEGF-B polypeptide. Preferred polynucleotides also include a promoter and/or enhancer to promote expression of the encoded VEGF-B protein in target cells of the recipient organism, as well as a stop codon, a polyadenylation signal sequence, and other sequences to facilitate expression. In a preferred embodiment, the VEGF-B product comprises an expression vector containing the VEGF-B-encoding polynucleotide. Viral vectors, such as replication-deficient adenoviral and adeno-associated viral vectors, retroviruses, lentiviruses and hybrids thereof, are preferred. In this and other embodiments, other growth factor-encoding polynucleotides may also be administered or co-administered using such vectors and expression modification elements.

In preferred embodiments, the composition that comprises the VEGF-B product further comprises a pharmaceutically acceptable carrier.

Other polypeptide factors that may modulate stem cell recruitment, proliferation, and differentiation are known, and may be co-administered with VEGF-B to enhance or modulate the recruitment, proliferation, and differentiation effects of VEGF-B. Thus, in one preferred variation, the method further comprises administering to the subject a myelopoietic agent selected from the group consisting of:

(a) granulocyte colony stimulating factor (G-CSF), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), interleukin-3 (IL-3), stem cell factor (SCF), vascular endothelial growth factor (VEGF or VEGF-A), vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), vascular endothelial growth factor E (VEGF-E),placental growth factor (PlGF) platelet derived growth factor A (PDGF-A), platelet derived growth factor B (PDGF-B), platelet derived growth factor C (PDGF-C), and platelet derived growth factor D (PDGF-D), NZ2 VEGF, D1701 VEGF-like protein, NZ10 VEGF-like protein (described in International Patent Application PCT/US99/25869), and fallotein;

(b) a polynucleotide comprising a nucleotide sequence encoding any member of (a), and

(c) combinations of one or more of these polypeptides or polynucleotides.

All of these growth factors have been described in literature, including the following:

Granulocyte colony stimulating factor (G-CSF), Swiss-Prot No. P09919, Nagata, et al., “Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor,” Nature 319:415-418(1986). G-CSF Genbank Acc. No.: S69115, Shimane, et al., “Molecular Cloning and Characterization of G-CSF Induced Gene cDNA,” Biochem. Biophys. Res. Commun., 199(1):26-32 (1994).

Interleukin-3 (IL-3), Swiss-Prot No. P26951, Kitamura, et al., “Expression cloning of the human IL-3 receptor cDNA reveals a shared beta subunit for the human IL-3 and GM-CSF receptors,” Cell, 66:1165-74(1991). IL-3, Gen Bank Acc. No. M33135, Phillips, et al., “Synthesis and expression of the gene encoding human interleukin-3,” Gene, 84(2):501-507 (1989).

Macrophage-CSF (M-CSF), Swiss-Prot No. P09603, Kawasaki, et al., “Molecular cloning of a complementary DNA encoding human macrophage-specific colony-stimulating factor (CSF-1),” Science 230:291-296(1985). M-CSF Genbank Acc. No. M64592, Cerretti, et al., “Human Macrophage-Colony Stimulating Factor: Alternative RNA and Protein Processing From a Single Gene,” Mol. Immunol. 25 (8):761-770 (1988).

Stem cell factor (SCF), Swiss Prot No: P21583, Martin, et al., “Primary structure and functional expression of rat and human stem cell factor DNAs,” Cell 63:203-211(1990). SCF, Genbank Acc. No. M59964, Martin, et al., “Primary Structure and Functional Expression of Rat and Human Stem Cell Factor DNAs,” Cell 63 (1):203-211 (1990).

Granulocyte-macrophage-CSF (GM-CSF), Swiss-Prot No.: PO4141, Lee et al., “Isolation of cDNA for a human granulocyte-macrophage colony-stimulating factor by functional expression in mammalian cells,” Proc. Natl. Acad. Sci. USA 82:4360-4364(1985).

Vascular endothelial growth factor (see e.g., GenBank Acc. No. Q16889 referred to herein for clarity as VEGF-A or by particular isoform), Swiss Prot No. P15692, Leung, et al., “Vascular endothelial growth factor is a secreted angiogenic mitogen,” Science 246:1306-09(1989). VEGF clone (a 581 bp cDNA covering bps 57-638, Genbank Acc. No. 15997). VEGF-A polynucleotide and polypeptide sequences are provided in SEQ ID NOS: 11 and 12 respectively.

Vascular endothelial growth factor C (VEGF-C), Swiss-Prot No.: P49767, Joukov, et al., “A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases,” EMBO. J. 15:290-298(1996), EMBO J. 15:1751-1751(1996). VEGF-C (see e.g., GenBank Acc. No. X94216; also known as VEGF related protein (VRP)). VEGF-C cDNA insert (Genbank Acc. No. X94216), see also U.S. Pat. No. 6,361,946. VEGF-C polynucleotide and polypeptide sequences are provided in SEQ ID NOS: 13 and 14 respectively.

Vascular endothelial growth factor D (VEGF-D (also known as c-fos-induced growth factor (FIGF); see e.g., Genbank Acc. No. AJ000185)), Swiss-Prot No.: 043915, Yamada et al., “Molecular cloning of a novel vascular endothelial growth factor, VEGF-D,” Genomics 42:483-488(1997). VEGF-D, Gen Bank Acc. No. D89630, Yamada et al., “Molecular Cloning of a Novel Vascular Endothelial Growth Factor, VEGF-D,” Genomics, 42(3):483-488 (1997). VEGF-D polynucleotide and polypeptide sequences are provided in SEQ ID NOS: 17 and 18 respectively.

Vascular endothelial growth factor E (VEGF-E) (also known as NZ7 VEGF or OV NZ7; see e.g., GenBank Acc. No. S67522). VEGF-E polynucleotide and polypeptide sequences are provided in SEQ ID NOS: 19 and 20 respectively.

Placental growth factor (PlGF), Maglione et al., Proc. Natl. Acad. Sci. USA, 88(20):9267-71 (1996) (PlGF, GenBank Acc. No. X54936). PlGF polynucleotide and polypeptide sequences are provided in SEQ ID NOS: 15 and 16 respectively.

Platelet-derived growth factors such as: Platelet-derived growth factor A (PDGF-A) (see e.g., GenBank Acc. No. X06374). PDGF-A polynucleotide and polypeptide sequences are provided in SEQ ID NOS: 23 and 24 respectively. Platelet-derived growth factor B (PDGF-B) (see e.g., GenBank Acc. No. M12783). PDGF-B polynucleotide and polypeptide sequences are provided in SEQ ID NOS: 25 and 26 respectively. Platelet-derived growth factor C (PDGF-C) polynucleotide and polypeptide sequences are provided in SEQ ID NOS: 6 and 7 respectively. Platelet-derived growth factor D (PDGF-D) polynucleotide and polypeptide sequences are provided in SEQ ID NOS: 8 and 9 respectively.

Other VEGF/PDGF family members or molecules having homology thereto such as: NZ2 VEGF (also known as OV NZ2; see e.g., GenBank Acc. No. S67520). NZ2 VEGF polynucleotide and polypeptide sequences are provided in SEQ ID NOS: 21 and 22 respectively. DI 701 VEGF-like protein (see e.g., GenBank Acc. No. AF106020; Meyer et al., EMBO J. 18:363-374). D1701 VEGF-like polynucleotide and polypeptide sequences are provided in SEQ ID NOS: 29 and 30 respectively. NZ10 VEGF-like protein (described in International Patent Application PCT/US99/25869) [Stacker and Achen, Growth Factors 17:1-11 (1999); Neufeld et al., FASEB J 13:9-22 (1999); Ferrara, J Mol Med 77:527-543 (1999)]. Fallotein, disclosed in the EMBL database (Acc. No. AF091434), which has structural characteristics of the PDGF/VEGF family of growth factors. Fallotein polynucleotide and polypeptide sequences are provided in SEQ ID NOS: 27 and 28 respectively.

The above-listed growth factors are not intended to be an exhaustive list. Use of any growth factor that can stimulate stem (progenitor) cells are contemplated as part of the present invention.

For the purposes of practicing the invention, biologically active fragments of these polypeptides and variants (e.g., variants with at least 90 or 95% identity to a wildtype form or an active fragment thereof) are considered equivalents of the polypeptides themselves. The same analysis applies with respect to polynucleotides encoding such polypeptides. Compositions, including those for use in manufacturing a medicament, comprising one or more growth factor products are also contemplated. The compositions used in the methods of the present invention are themselves considered to be part of the invention.

Another embodiment of the invention is a method of stimulating stem cell proliferation or differentiation, comprising, obtaining a biological sample from a mammalian subject, wherein said sample comprises stem cells, and contacting the stem cells with a composition comprising a vascular endothelial growth factor B (VEGF-B) product. In this method, the beneficial effects of the VEGF-B are imparted to cells from a human or animal subject outside of the body of the human or other animal subject. Such therapy may be desirable to avoid VEGF-B side effects, or to prepare a treated cell sample for use in a medical procedure.

The biological sample can be any tissue or fluid sample from which stem cells are found. Blood and bone marrow are preferred sources for the biological sample, as is umbilical cord blood.

In a preferred embodiment, the biological sample is subjected to at least some purification and/or isolation procedures to purify or isolate the stem cells. For example, removal of red blood cells from a blood sample constitutes one level of purification/isolation. Still further purification, e.g., to select those nucleated cells that are CD34+ and/or VEGFR-1+, may be performed prior to the VEGF-B treatment. In a preferred embodiment, the purified stem cells comprise VEGFR-1+ or CD34+ or CD133+stem cells. Still more preferred are stem cells that comprise two or more of these markers.

Likewise, in some variations of the invention, it is desirable to purify or isolate the stem cells after the VEGF-B treatment to select those cells that have proliferated or differentiated in response to the VEGF-B treatment.

In one variation, the contacting step comprises culturing the stem cells in a culture containing the VEGF-B product. 1-10 μg protein/ml growth medium will give maximum growth stimulation. In still another variation, the contacting comprises transforming or transfecting the stem cells with a VEGF-B transgene.

In preferred variations, the method further comprises a step of returning the stem cells to the mammalian subject from which they were originally removed. Alternatively, the method comprises a step of transplanting the cells into a different mammalian subject. Human subjects are preferred. In preferred embodiments, where the cell donor is a close relative, or has a substantially identical human leukocyte antigen (HLA) profile.

Such ex vivo therapy is useful in a variety of contexts. For example, with a human subject that needs antineoplastic radiation or chemotherapy, healthy stem cells can be removed prior to the radiation or chemotherapy, cultured according to the invention, and returned following the radiation or chemotherapy. Thus, the biological sample is obtained prior to administering a dose of chemotherapy or radiation, and the stem cells are returned to the human subject after the contacting step and after the dose of chemotherapy or radiation.

The method also is useful for autologous or heterologous bone marrow transplantation. Similarly, the stem cells treated according to the method of the invention are expected to improve the success and reduce side effects of organ or tissue transplantation and graft attachments. In one variation, the cells are seeded into a tissue, organ, or artificial matrix ex vivo, and said tissue, organ, or artificial matrix is attached, implanted, or transplanted into the mammalian subject.

The term “VEGF-B product” for this embodiment of the invention has the same meaning set forth above.

The beneficial effects of contacting the cells with VEGF-B can be further enhanced by contacting the cells with one or more additional myelopoietic agents, as described above. These additional agents can be used contemporaneously with the VEGF-B product or serially, in any order.

In ex vivo embodiments, active forms of particular growth factor(s) are preferred for contacting the cells. For example, proteins that are naturally synthesized as pre-proteins, prepro-proteins, or other pre-modified forms that are not fully active are preferably administered in processed or modified forms that are active. Polynucleotides for use in ex vivo therapy are preferably manipulated to produce a “recombinantly processed” form of polypeptide by removal—at the polynucleotide level—of sequences that encode pro-peptides or other domains whose removal is required for optimal activity. Alternatively, if inactive growth factors are applied, then agents may be co-administered that result in the activation of the growth factor.

For example, a protease can be co-administered with PDGF-C in order to cleave the CUB domain and activate the protein. “Activation” is understood as a processing of a growth factor so that it is able to bind to and/or activate a receptor of the growth factor.

Another embodiment of the invention provides a method of stimulating stem cell recruitment, proliferation, or differentiation comprising, identifying a human subject in need of stem cell recruitment, proliferation, or differentiation, and administering to the human subject a composition comprising a platelet derived growth factor (PDGF) product. This embodiment and its numerous variations are similar to an embodiment described above with respect to VEGF-B, except that a PDGF product is employed in this embodiment. As described herein in detail, the biological activities of PDGFs, expecially PDGF-C, make such methods particularly useful for treating ischemic conditions.

In one variation the PDGF product comprises a PDGF polypeptide. Naturally occurring PDGF polypeptides are preferred, and human PDGF polypeptides are highly preferred. At least four distinct PDGF family members have been identified, PDGF-A, PDGF-B, PDGF-C, and PDGF-D.

PDGF-A and PDGF-B were characterized first in the literature and have thus been the subject of a greater body of research and development. Homo- and heterodimers have been formed with these polypeptides, and variants have been described with altered amino acid sequences yet the same or similar receptor binding properties. Exemplary PDGF-A and -B polypeptides for use in the invention have been described in U.S. Pat. Nos. 5,605,816 (PDGF-A and A/B heterodimers); 4,889,919 (PDGF-A homodimers); 5,759,815 (recombinant production of PDGF-A or -B in prokaryotes and formation of various dimers); 5,889,149 (PDGF-AB isoforms); 4,845,075 and 5,428,010 and 5,516,896 (PDGF-BB homodimers); 5,272,064 and 5,512,545 (PDGF-B analogues); 5,905,142 (protease-resistant PDGF-B analogues); and 5,128,321 and 5,498,600 and 5,474,982 (PDGF-A/B mosaics). In addition to the foregoing patent documents, there is substantial scientific literature describing and characterizing PDGF-A and -B proteins.

In a preferred embodiment, the PDGF polypeptide comprises a PDGF-C or PDGF-D polypeptide. PDGF-C polypeptides and polynucleotides were characterized by Eriksson et al. in International Patent Publication No. WO 00/18212, U.S. Patent Application Publication No. 2002/0164687 A1, and U.S. patent application Ser. No. 10/303,997 [published as U.S. Pat. Publ. No. 2003/0211994]. PDGF-D polynucleotides and polypeptides were characterized by Eriksson, et al. in International Patent Publication No. WO 00/27879 and U.S. Patent Application Publication No. 2002/0164710 A1. These documents are all incorporated by reference in their entirety. As described therein, PDGF-C and -D bind to PDGF receptors alpha and beta, respectively. However, a noteworthy distinction between these polypeptides and PDGF-A and -B is that PDGF-C and -D each possess an amino-terminal CUB domain that can be proteolytically cleaved to yield a biologically active (receptor binding) carboxy-terminal domain with sequence homology to other PDGF family members. For convenience, exemplary PDGF-C and -D polynucleotide and deduced amino acid sequences have been appended hereto as SEQ ID NOS: 6-9.

A preferred form of PDGF-C comprises the PDGF/VEGF homology domain (PVHD) of PDGF-C and retains receptor binding and activation functions. The minimal domain is approximately residues 230-345 of SEQ ID NO: 7. However, the domain can extend towards the N terminus up to residue 164. The PVHD of PDGF-C is also referred to as truncated PDGF-C. The truncated PDGF-C is an activated form of PDGF-C. A putative proteolytic site in PDGF-C is found in residues 231-234 of SEQ ID NO: 7, a dibasic motif. The putative proteolytic site is also found in PDGF-A, PDGF-B, VEGF-C and VEGF-D. In these four proteins, the putative proteolytic site is also found just before the minimal domain for the PDGF/VEGF homology domain. The CUB domain of PDGF-C represents approximately amino acid residues 23-159 of SEQ ID NO: 7. U.S. Patent Application Publication No.: 2002/0164687.

Similar to PDGF-C, PDGF-D has a two domain structure with a N-terminal CUB domain (described as approximately residues 67-167 or 54-171 of SEQ ID NO: 9) and a C-terminal PDGF/VEGF homology domain (PVHD). A putative proteolytic site in PDGF-D is found in residues 255-258 of SEQ ID NO: 9. A preferred PDGF-D polypeptide comprises the PDGF/VEGF homology domain (PVHD) of PDGF-D and retains receptor binding and activation functions. The minimal domain of PDGF-D is approximately residues 272-362 or 255-370 of SEQ ID NO: 9. However, PDGF-D's PVHD extends toward the N terminus up to residue 235 of SEQ ID NO: 9. The truncated PDGF-D is the putative activated form of PDGF-D. U.S. Patent Application Publication No. 2002/0164710.

As discussed elsewhere herein in detail, members of the PDGF/VEGF family form homodimers (and sometimes heterodimers). References herein to specific dimeric forms (e.g., PDGF-CC for PDGF-C homodimers) is sometimes made for context or clarity. References to polypeptide forms (e.g. PDGF-C) are not meant to imply anything about the monomeric or dimeric or other forms of the polypeptide composition, unless specifically stated.

In addition to naturally occurring PDGF polypeptides, variant forms that still bind to and/or the respective PDGF receptors (including receptor homodimers and heterodimers) also may be used in the invention described herein. Variants with at least 90% amino acid sequence identity to a naturally occurring human PDGF-A -B, -C, or -D polypeptide are preferred. Still more preferred is at least 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Thus, in another variation, the PDGF polypeptide comprises a portion of the amino acid sequence set forth in SEQ ID NOS: 24, 26, 7, or 9 that is effective to bind PDGFR-α and/or PDGFR-β. In still another variation, the PDGF polypeptide binds PDGFR-α and/or PDGFR-β and is encoded by a polynucleotide that hybridizes under stringent conditions with the complement of the polynucleotide in SEQ ID NO: 23, 25, 6, or 8.

In some embodiments, the PDGF-C polypeptide has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to residues 230-345 of SEQ ID NO: 7. In some embodiments, the PDGF-D polypeptide has at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to residues 272-362 of SEQ ID NO: 9.

In another variation, the PDGF product comprises a polynucleotide that encodes a PDGF polypeptide. Expression of the polynucleotide in or near target progenitor cells results in production of effective quantities of PDGF polypeptides. Thus, in preferred variations, the PDGF product comprises a vector, such as a viral vector, containing the polynucleotide. Exemplary vectors include replication-deficient adenoviral or adeno-associated viral vectors, as well as retroviruses and lentiviruses. However, any vector effective for delivery of a PDGF polynucleotide to target cells is contemplated.

In still another variation, the PDGF product is co-administered with one or more additional myelopoietic agents which together stimulate recruitment, proliferation, and/or differentiation of the target cells in a desirable way. Exemplary agents for coadministration with a PDGF product include those growth factors and other agents described earlier in respect to VEGF-B therapies, as is coadministration with VEGF-B. Such growth factors include: (a) granulocyte colony stimulating factor (G-CSF), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), interleukin-3 (IL-3), stem cell factor (SCF), vascular endothelial growth factor (VEGF), vascular endothelial growth factor B (VEGF-B) vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), and placental growth factor (PlGF); (b) a polynucleotide comprising a nucleotide sequence encoding any member of (a), and (c) combinations thereof. Other growth factors not listed may also be employed.

The present invention is also based on the discovery that PDGF-CC (a PDGF-C dimer) enhances post-ischemic revascularization in the heart and limb, apparently exerting effects on vascular progenitor and mature cells of both endothelial and smooth muscle cell/fibroblast lineages. Revascularization of brain, heart, limb, and other tissues that have become ischemic are contemplated. As described herein in detail, evidence indicates that PDGF-CC mobilizes endothelial progenitor cells, induces differentiation of bone marrow cells into endothelial cells, stimulates migration of endothelial cells, and upregulates VEGF expression. Moreover, PDGF-CC induces the differentiation of bone marrow cells into smooth muscle cells (SMC) and stimulates SMC growth and migration during vessel sprouting. This pleiotropic activity of PDGF-CC on vascular progenitors and differentiated cells of both endothelial and smooth muscle cell/fibroblast lineages together with evidence of its safety profile (lack of hemangioma-genesis, edema or fibrosis), and evidence that certain activity is restricted to ischemic conditions, provides novel therapeutic indications for PDGF-C products in vivo and ex vivo for treating ischemic diseases.

One embodiment includes a method of stimulating stem cell proliferation or differentiation. A biological sample comprising stems cells is obtained from a mammalian subject, wherein said sample comprises stem cells. The stem cells are then contacted with a composition comprising a platelet derived growth factor-C (PDGF-C) product. In one variation stem cells are isolated from the biological sample prior to the contacting step, including variation wherein AC133+/CD34+ cells are isolated from the biological sample. In some variations, stems cells are contacted with the PDGF-C product until particular cell surface markers appear (become more prominent in) and/or particular markers disappear from (become less prominent in) a stem cell population. In one variation, the contacting continues until stem cells differentiate into CD144+cells, at which time PDGF-C treatment is stopped. In another variation, the contacting continues until stem cells differentiate into SMA+/CD 144−/CD31−/CD34− cells. In some embodiments, a VEGF-A product in addition to a PDGF-C product is used to contact the cells.

The PDGF-C products contemplated from practice of the foregoing method include PDGF-C polypeptides and polynucleotides that encode them, or combination thereof. Where the PDGF-C product comprises a PDGF-C polypeptide, the PDGF-C polypeptide preferably comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 10 and binds to either a PDGFR-α/α homodimer or PDGFR-α/β heterodimer receptor. In one embodiment, the polypeptide is present in the composition as homodimers (PDGF-CC). In some variations, the PDGF-C polypeptide is encoded by a polynucleotide that hybridizes under stringent conditions with the complement of the polynucleotide in SEQ ID NO: 6.

One embodiment of ex vivo therapy of the invention involves separate treatment of two or more aliquots of progenitor cells from a patient with two or more distinct growth factor regimens of one or more growth factors per regimen. In this way, differentiation into two or more distinct populations of cells is achieved. These distinct cell populations preferably complement each other in vivo to achieve improved therapeutic benefit when readministered to a patient. In one embodiment, stem cell proliferation or differentiation is carried out by first obtaining a biological sample from a mammalian subject, wherein said sample comprises stem cells. A first aliquot of the stem cells is contacted with a first composition comprising a first growth factor product selected from a VEGF-B product and PDGF-C product. A second aliquot of the stem cells with a second composition comprising a second growth factor product independently selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PDGF-A, PDGF-B, PDGF-C, and PlGF products. In such an embodiment, the first and second growth factor products are generally not the same. In some variations, the first growth factor product is a PDGF-C product and the second growth factor product is a VEGF-A product.

In another embodiment, the differentiation of stem cells into both endothelial and smooth muscle cells is promoted by obtaining a biological sample comprising stem cells from a mammalian subject, wherein said sample comprises stem cells. The obtained cells are then contacted with a composition comprising a platelet-derived growth factor-C (PDGF-C) product, in an amount and for a time sufficient to cause the cells to differentiate into both endothelial and smooth muscle cells. In some variations, the contacted cells are returned to the mammalian subject. In some variations, the mammalian subject who receives the cells has an ischemic condition, including one affecting tissue of the heart (e.g., infarction), brain (e.g., stroke) or limb (e.g., peripheral clot).

In another embodiment, an ischemic condition is ameliorated by first (optionally) diagnosing a mammalian subject with an ischemic condition. A biological sample comprising stem cells is obtained from a subject so diagnosed. The obtained cells are contacted with a composition comprising a platelet-derived growth factor-C (PDGF-C) product, in an amount and for a time sufficient to cause the cells to differentiate into both endothelial and smooth muscle cells. The contacted cells are then returned to the mammalian subject. In some embodiments, the cells are returned by implanting or injecting the cells into ischemic tissue of the mammalian subject.

Still another embodiment of the invention is a method of stimulating stem cell proliferation or differentiation, comprising obtaining a biological sample from a mammalian subject, wherein said sample comprises stem cells, and contacting the stem cells with a composition comprising a platelet derived growth factor (PDGF) product. This method and its variations are similar to an embodiment summarized above with respect to VEGF-B. In a preferred variation, the contacting comprises culturing the stem cells in a culture containing the PDGF product.

In one variation, the stem cells are purified and isolated after obtaining the sample and before contacting them with the PDGF product. In one variation, the stem cells are purified and isolated after treatment with the PDGF product in the contacting step. Preferred populations of stem cells for purification include those expressing one or more of the following receptors/markers on their cell surface: PDGFR-alpha, PDGFR-beta, and CD34.

Progenitor/stem cells that have been prepared according to the various ex vivo embodiments of the invention are useful in a number of therapeutic contexts when returned to the host from which the sample was originally obtained, or transplanted into a different host. The cells can be returned into the bloodstream or bone marrow intravenously or by injection, or alternatively, seeded into a tissue, organ, or artificial matrix ex vivo, and said tissue, organ, or artificial matrix is attached, implanted, or transplanted into the mammalian subject.

In one preferred embodiment, the cells are used to treat a human subject that needs antineoplastic chemotherapy. For example, the biological sample is obtained prior to administering a dose of chemotherapy, and the stem cells are returned to the human subject after the contacting and after the dose of chemotherapy.

In one preferred embodiment, the cells are used to treat a human subject who has been diagnosed with a cardiovascular diseases, including diabetes-related vascular complications. Human subjects suffereing from either Type I (insulin-dependent) or Type II (non-insulin-dependent) diabetes mellitus are contemplated, as are non-diabetic human subjects who suffer from cardiovascular diseases. Millions of patients suffer from insufficient blood supply to tissues, particularly to the heart, brain and legs. The growing population of diabetics is particularly prone to developing these life-threatening conditions, as are those of risk of heart attacks and strokes. Therapeutic angio/arteriogenic factors are therefore of interest for alleviating such complications by inducing new blood vessels. The building of new stable and functional vessels relies on a concerted action of vascular progenitors and differentiated endothelial and smooth muscle cells. Therapeutic angiogenesis may thus require co-administration of factors that affect both lineages. Alternatively, molecules with pleiotropic effects on both lineages would be attractive, but only a few have been identified thus far.

In other therapeutic contexts, it may be desirable to suppress progenitor/stem cell recruitment, proliferation, or differentiation. Further embodiments of the invention are methods of inhibiting/suppressing progenitor/stem cell recruitment, proliferation, or differentiation by contacting the cells (in vivo or ex vivo) with inhibitors specific for the VEGF-B or the PDGF products described above. Exemplary inhibitors, including antibodies, antisense molecules, and aptamers, are described in greater detail below.

It will be apparent that many aspects of the invention relate to new uses of various polynucleotide and protein products. In still another variation, the invention provides for the use of any of the aforementioned products in the manufacture of a medicament for stimulating stem cell recruitment, proliferation, and/or differentiation, or a medicament for treatment of any disease or condition that would benefit from stem cell recruitment, proliferation, and/or differentiation.

Likewise, the specific inhibitors described above are useful in the manufacture of a medicament for inhibition of stem cell recruitment, proliferation, and/or differentiation, or a medicament for treatment of any disease or condition that would benefit (even transiently) from inhibition of stem cell recruitment, proliferation, and/or differentiation.

Unitary activity on a single type of cell leading to nonfunctional capillaries, or harmful side effects involving edema or angioma-genesis, is often the central problem for therapeutic vasculogenic, angio/arteriogenic, and neoangiogenic agents under trial. Molecules with pleiotropic activities affecting multiple vascular cells or stages of vasculogenesis, angio/arteriogenesis, and neoangiogenesis, but with minimal side effects, thus become attractive means to treat tissue ischemia. There are considerable potential advantages of choosing such molecules, including mobilizing multiple vascular cells and molecules needed to build functional vessels by a single delivery of one effector molecule, and the simultaneous regulation of the complex cascade of vasculogenesis, angio/arteriogenesis, and/or neoangiogenesis with one therapeutic intervention. This represents a promising paradigm of new therapeutic agents to cultivate functional vessels with more physiological functional properties in treating tissue ischemia.

Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are intended as aspects of the invention.

Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only those limitations that are described herein as critical to the invention should be viewed as such; variations of the invention lacking features that have not been described herein as critical are intended as aspects of the invention.

With respect to aspects of the invention that have been described as a set or genus, every individual member of the set or genus is intended, individually, as an aspect of the invention.

In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. Although the applicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1[0120] a shows VEGFR-3⁺ cells (%) in blood of 5-fluorouracil treated FVB mice (n=4), relative to total white blood cells.
FIG. 1[0121] b shows VEGFR-3⁺ (%) cells in bone marrow after 5-FU treatment of FVB mice.
FIG. 2[0122] a shows white blood cell counts of blood in 5-FU and adenovirus treated mice.
FIG. 2[0123] b shows white blood cell counts of blood in 5-FU and adenovirus treated NMRI mice.
FIG. 2[0124] c shows white blood cell counts of blood after 5-FU and adenoviral treatment on day 10 in FVB mice.
FIG. 3[0125] a shows VEGFR-1⁺ cells (%) of white blood cells in nude mice treated with an adenovirus containing a LacZ, VEGF-C, VEGF-C156S or VEGF-B transgene.
FIG. 3[0126] b shows VEGFR-2⁺ cells (%) of white blood cells in nude mice treated with an adenovirus containing a LacZ, VEGF-C, VEGF-C 156S or VEGF-B transgene.
FIG. 3[0127] c shows VEGFR-3⁺ cells (%) of white blood cells in nude mice treated with an adenovirus containing a LacZ, VEGF-C, VEGF-C156S or VEGF-B transgene.
FIG. 3[0128] d shows CD34⁺ cells (%) of white blood cells in nude mice treated with an adenovirus containing a LacZ, VEGF-C, VEGF-CI 56S or VEGF-B transgene.
FIG. 4 shows the number of endothelial progenitor cells per square millimeter in mice either treated with a control or PDGF-CC. There were three sets of control and experimental animals: non-ischemic mice, ischemic mice sacrificed after two days and ischemic mice sacrificed after five days. [0129]
FIG. 5 shows results of bone marrow cell adherence assays of control cells, cells treated with VEGF and cells treated with PDGF-CC. [0130]
FIG. 6[0131] a shows migration assay results of three types of cells—bovine aortic endothelial cells (BAEC), human microvascular endothelial cells (HMVEC) and smooth vessel cells (SMC)—and how that migration was influenced by the absence or presence of various growth factors—VEGF, PDGF-AA, PDGF-BB, and PDGF-CC.
FIG. 6[0132] b shows proliferation assay results of HMVEC influenced by the absence or presence of various growth factors—VEGF, PDGF-AA, PDGF-BB, and PDGF-CC.
FIG. 7[0133] a shows the results from aortic ring assays, and specifically microvessel outgrowth influenced by the absence or presence of various growth factors—VEGF, PDGF-AA, PDGF-BB, and PDGF-CC.
FIG. 7[0134] b shows the results from aortic ring assays, and specifically fibroblast proliferation and migration influenced by the absence or presence of various growth factors—VEGF, PDGF-AA, PDGF-BB, and PDGF-CC.
FIG. 8[0135] a shows in an upper panel a Western blot of protein immunoprecipitated from lysates of human smooth muscle cells (hSMC) and NIH-3T3 cells (a fibroblast cell line) using an anti-PDGFR-α antibody. In a lower panel a Western blot of proteins from human smooth muscle cells (hSMC) and NIH-3T3 cells using an anti-phosphotyrosine antibody is shown.
FIG. 8[0136] b shows proliferation of NIH-3T3 cells and hSMC cells influenced by various PDGF homodimers.
FIG. 8[0137] c shows on the left-hand side the results from both RNA protection assays (RPA) (top panel) and a Western blot (bottom panel) of either control (vector) NIH-3T3 cells or NIH-3T3 cells overexpressing PDGF-C. The right hand shows the results of ELISAs of either control (vector) NIH-3T3 cells or NIH-3T3 cells overexpressing PDGF-C.

DETAILED DESCRIPTION

The term “VEGF-B” as used in the present invention encompasses those polypeptides identified as VEGF-B in U.S. Pat. No. 6,331,301, which is incorporated herein in its entirety, as well as published U.S. Application No. 2003/0008824. [0138]
VEGF-B comprises, but is not limited to, both the VEGF-B[0139] ₁₆₇and/or VEGF-B₁₈₆isoforms or a fragment or analog thereof having the ability to bind VEGFR-1. Active analogs should exhibit at least 85% sequence identity, preferably at least 90% sequence identity, particularly preferably at least 95% sequence identity, and especially preferably at least 98% sequence identity to the natural VEGF-B polypeptides, as determined by BLAST analysis. The active substance typically will include the amino acid sequence Pro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys (where Xaa may be any amino acid) (SEQ ID NO: 5) that is characteristic of VEGF-B.
Use of polypeptides comprising VEGF-B sequences modified with conservative substitutions, insertions, and/or deletions, but which still retain the biological activity of VEGF-B is within the scope of the invention. Standard methods can readily be used to generate such polypeptides including site-directed mutagenesis of VEGF-B polynucleotides, or specific enzymatic cleavage and ligation. Similarly, use of peptidomimetic compounds or compounds in which one or more amino acid residues are replaced by a non-naturally-occurring amino acid or an amino acid analog that retains the required aspects of the biological activity of VEGF-B is contemplated. [0140]
The term PDGF comprises, but is not limited to PDGF-A, PDGF-B, PDGF-C, and PDGF-D, or a fragment or analog thereof having the ability to bind PDGF-receptors. PDGF-A may bind to and/or activate PDGFR-α/α homodimers. PDGF-β may bind to and/or activate PDGFR-α/α homodimers, PDGFR-α/β heterodimers and PDGR-β/β homodimers. PDGF-C may bind to and/or activate PDGFR-α/α homodimers. PDGF-C may also bind to and/or activate PDGFR-α/β heterodimers via PDGFR-α binding. PDGF-D may bind to and/or activate PDGFR-β/β homodimers. PDGF-D may also bind to and/or activate PDGFR-α/β heterodimers via PDGFR-β binding. Active analogs should exhibit at least 85% sequence identity, preferably at least 90% sequence identity, particularly preferable at least 95% sequence identity, and especially preferable at least 98% sequence identity to the natural PDGF polypeptides, as determined by BLAST analysis. [0141]
Use of polypeptides comprising PDGF sequences modified with conservative substitutions, insertions, and/or deletions, but which still retain the biological activity of PDGFs is within the scope of the invention. Standard methods can readily be used to generate such polypeptides including site-directed mutagenesis of PDGF polynucleotides, or specific enzymatic cleavage and ligation. Similarly, use of peptidomimetic compounds or compounds in which one or more amino acid residues are replaced by a non-naturally-occurring amino acid or an amino acid analog that retains the required aspects of the biological activity of PDGFs is contemplated. [0142]
In addition, variant forms of VEGF-B or PDGF polypeptides that may result from alternative splicing and naturally-occurring allelic variation of the nucleic acid sequence encoding VEGF-B or a PDGF are useful in the invention. Allelic variants are well known in the art, and represent alternative forms or a nucleic acid sequence that comprise substitution, deletion or addition of one or more nucleotides, but which do not result in any substantial functional alteration of the encoded polypeptide. [0143]
Variant forms of VEGF-B or a PDGF can be prepared by targeting non-essential regions of a VEGF-B or PDGF polypeptide for modification. These non-essential regions are expected to fall outside the strongly-conserved regions of the VEGF/PDGF family of growth factors. In particular, the growth factors of the PDGF/VEGF family, including VEGF-B and the PDGFs, are dimeric, and at least VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-A and PDGF-B show complete conservation of eight cysteine residues in the N-terminal domains, i.e. the PDGF/VEGF-like domains. [Olofsson, et al., [0144] Proc. Nat'l. Acad. Sci. USA, 93:2576-2581 (1996); Joukov, et al., EMBO J., 15:290-298 (1996).] These cysteines are thought to be involved in intra- and inter-molecular disulfide bonding. In addition there are further strongly, but not completely, conserved cysteine residues in the C-terminal domains. Loops 1, 2 and 3 of each subunit, which are formed by intra-molecular disulfide bonding, are involved in binding to the receptors for the PDGF/VEGF family of growth factors. [Andersson, et al., Growth Factors, 12:159-64 (1995).]
These conserved cysteine residues are preferably preserved in any proposed variant form, although there may be exceptions, because receptor-binding VEGF-B analogs are known in which one or more of the cysteines is not conserved. Similarly, the active sites present in [0145] loops 1, 2 and 3 also should be preserved. Other regions of the molecule can be expected to be of lesser importance for biological function, and therefore offer suitable targets for modification. Modified polypeptides can readily be tested for their ability to show the biological activity of VEGF-B or a PDGF by routine activity assay procedures such as a VEGFR-1 binding assay or a stem cell proliferation assay based on the examples set forth below.
Preferably, where amino acid substitution is used, the substitution is conservative, i.e. an amino acid is replaced by one of similar size and with similar charge properties. [0146]
As used herein, the term “conservative substitution” denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like. Neutral hydrophilic amino acids that can be substituted for one another include asparagine, glutamine, serine and threonine. The term “conservative substitution” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. [0147]
Alternatively, conservative amino acids can be grouped as described in Lehninger, ([0148] Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY, pp. 71-77 (1975)) as set out in the following: Non-polar (hydrophobic) A. Aliphatic: A, L, I, V, P, B. Aromatic: F, W, C. Sulfur-containing: M, D. Borderline: G. Uncharged-polar A. Hydroxyl: S, T, Y, B. Amides: N, Q, C. Sulfhydryl: C, D. Borderline: G. Positively Charged (Basic): K, R, H. Negatively Charged (Acidic): D, E.
VEGF-B or a PDGF protein can be modified, for instance, by glycosylation, amidation, carboxylation, or phosphorylation, or by the creation of acid addition salts, amides, esters, in particular C-terminal esters, and N-acyl derivatives. The proteins also can be modified to create peptide derivatives by forming covalent or noncovalent complexes with other moieties. Covalently bound complexes can be prepared by linking the chemical moieties to functional groups on the side chains of amino acids comprising the peptides, or at the N- or C-terminus. [0149]
VEGF-B and PDGF proteins can be conjugated to a reporter group, including, but not limited to a radiolabel, a fluorescent label, an enzyme (e.g., that catalyzes a calorimetric or fluorometric reaction), a substrate, a solid matrix, or a carrier (e.g., biotin or avidin). [0150]
Examples of VEGF-B analogs are described in WO 98/28621 and in Olofsson, et al., [0151] Proc. Nat'l. Acad. Sci. USA, 95:11709-11714 (1998), both incorporated herein by reference. Examples of PDGF analogs are described in U.S. Pat. Nos. 5,512,545, and 5,474,982; U.S. Patent Application Nos.: 20020164687 and 20020164710.
VEGF-B and PDGF polypeptides are preferably produced by expression of DNA sequences that encode them such as DNAs that correspond to, or that hybridize under stringent conditions with the compliments of SEQ ID NOS: 1 and 3. Suitable hybridization conditions include, for example, 50% formamide, 5×SSPE buffer, 5× Denhardts solution, 0.5% SDS and 100 μg/ml of salmon sperm DNA at 42° C. overnight, followed by washing 2×30 minutes in 2×SSC at 55° C. Such hybridization conditions are applicable to any polynucleotide encoding one or more of the growth factors of the present invention. [0152]
The invention is also directed to an isolated and/or purified DNA that corresponds to, or that hybridizes under stringent conditions with, any one of the foregoing DNA sequences. [0153]
The VEGF-B proteins and polypeptides for use in the present invention are characterized by the amino acid sequence Pro-Xaa-Cys-Val-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys (SEQ ID NO: 5) and having the property of stimulating the recruitment, mobilization or proliferation of stem cells, including hematopoietic progenitor cells and endothelial progenitor cells, wherein the protein comprises a sequence of amino acids substantially corresponding to an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NOS: 2 and 4. VEGF-B dimmers may comprise VEGF-B polypeptides of identical sequence, of different VEGF-β isoforms, or other heterogeneous VEGF-B molecules. [0154]
The VEGF-B for use according to the present invention can be used in the form of a protein dimer comprising VEGF-B protein, particularly a disulfide-linked dimer. The protein dimers of the invention include both homodimers of VEGF-B and heterodimers of VEGF-B and VEGF polypeptides, as well as other VEGF family growth factors including, but not limited to placental growth factor (PlGF), which are capable of binding to VEGFR-1 (fit-1). The VEGF-B of the present invention also includes VEGF-B polypeptides that have been engineered to contain a N-glycosylation cite such as those described in Jeltsch, et al., WO 02/07514, which is incorporated herein in its entirety. [0155]
As used herein, the term “biologically active,” when used in conjunction with VEGF-B refers to a VEGF-B polypeptide that binds VEGFR-1 (also known as flt-1) in a manner substantially similar to that of full length VEGF-B, and/or that stimulates migration, proliferation and/or differentiation of a population of mammalian stem cells. As used herein, the term biologically active,” when used in conjunction with PDGF refers to a PDGF polypeptide that binds to its natural PDGF-receptor (PDGF-α and/or PDGF-β as described above) in a manner substantially similar to that of the native PDGF, and/or that stimulates migration, proliferation and/or differentiation of a population of a mammalian stem cells. [0156]
The term “vector” refers to a nucleic acid molecule amplification, replication, and/or expression vehicle in the form of a plasmid or viral DNA system where the plasmid or viral DNA may be functional with bacterial, yeast, invertebrate, and/or mammalian host cells. The vector may remain independent of host cell genomic DNA or may integrate in whole or in part with the genomic DNA. The vector will contain all necessary elements so as to be functional in any host cell it is compatible with. Such elements are set forth below. [0157]
Preparation of VEGF-B is discussed in U.S. Pat. No. 6,331,301, which is incorporated herein in its entirety. [0158]
Preparation of DNA Encoding VEGF-B or PDGF Polypeptides [0159]
A nucleic acid molecule encoding VEGF-B or PDGF can readily be obtained in a variety of ways, including, without limitation, chemical synthesis, cDNA or genomic library screening, expression library screening, and/or PCR amplification of cDNA. These methods and others useful for isolating such DNA are set forth, for example, by Sambrook, et al., “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), by Ausubel, et al., eds., “Current Protocols In Molecular Biology,” Current Protocols Press (1994), and by Berger and Kimmel, “Methods In Enzymology: Guide To Molecular Cloning Techniques,” vol. 152, Academic Press, Inc., San Diego, Calif. (1987). Preferred nucleic acid sequences encoding VEGF-B or PDGF are mammalian sequences. Most preferred nucleic acid sequences encoding VEGF-B or PDGF are human, rat, and mouse. [0160]
Chemical synthesis of a VEGF-B or PDGF nucleic acid molecule can be accomplished using methods well known in the art, such as those set forth by Engels, et al., [0161] Angew. Chem. Intl. Ed., 28:716-734 (1989). These methods include, inter alia, the phosphotriester, phosphoramidite and H-phosphonate methods of nucleic acid synthesis. Typically, the nucleic acid molecule encoding the full length VEGF-B polypeptide will be several hundred base pairs (bp) or nucleotides in length. Nucleic acids larger than about 100 nucleotides in length can be synthesized as several fragments, each fragment being up to about 100 nucleotides in length. The fragments can then be ligated together, as described below, to form a full length nucleic acid encoding the VEGF-B or PDGF polypeptide. A preferred method is polymer-supported synthesis using standard phosphoramidite chemistry.
Alternatively, the nucleic acid encoding a VEGF-B or PDGF polypeptide may be obtained by screening an appropriate cDNA library prepared from one or more tissue source(s) that express the polypeptide, or a genomic library from any subspecies. The source of the genomic library may be any tissue or tissues from any mammalian or other species believed to harbor a gene encoding VEGF-B or a VEGF-B homologue or PDGF or PDGF homologue. [0162]
The library can be screened for the presence of the VEGF-B cDNA/gene using one or more nucleic acid probes (oligonucleotides, cDNA or genomic DNA fragments that possess an acceptable level of homology to the VEGF-B or VEGF-B homologue cDNA or gene to be cloned) that will hybridize selectively with VEGF-B or VEGF-B homologue cDNA(s) or gene(s) that is(are) present in the library. The probes preferably are complementary to or encode a small region of the VEGF-B DNA sequence from the same or a similar species as the species from which the library was prepared. Alternatively, the probes may be degenerate, as discussed below. [0163]
The library also can be screened for the presence of the PDGF cDNA/gene using one or more nucleic acid probes (oligonucleotides, cDNA or genomic DNA fragments that possess an acceptable level of homology to the PDGF homologue cDNA or gene to be cloned) that will hybridize selectively with PDGF or PDGF homologue cDNA(s) or gene(s) that is(are) present in the library. The probes preferably are complementary to or encode a small region of the PDGF DNA sequence from the same or a similar species as the species from which the library was prepared. Alternatively, the probes may be degenerate, as discussed below. [0164]
Where DNA fragments (such as cDNAs) are used as probes, typical hybridization conditions are those for example as set forth in Ausubel, et al., eds., supra. After hybridization, the blot containing the library is washed at a suitable stringency, depending on several factors such as probe size, expected homology of probe to clone, type of library being screened, number of clones being screened, and the like. Examples of stringent washing solutions (which are usually low in ionic strength and are used at relatively high temperatures) are as follows. One such stringent wash is 0.015 M NaCl, 0.005 M NaCitrate and 0.1 percent SDS at 55-65° C. Another such stringent buffer is 1 mM Na[0165] ₂EDTA, 40 mM NaHPO₄, pH 7.2, and 1 percent SDS at about 40-50° C. One other stringent wash is 0.2.×SSC and 0.1 percent SDS at about 50-65° C. Such hybridization conditions are applicable to any polynucleotide encoding one or more of the growth factors of the present invention.
Another suitable method for obtaining a nucleic acid encoding a VEGF-B or PDGF polypeptide is the polymerase chain reaction (PCR). In this method, poly(A)+RNA or total RNA is extracted from a tissue that expresses VEGF-B or PDGF (such as lymphoid tissue). cDNA is then prepared from the RNA using the enzyme reverse transcriptase. Two primers typically complementary to two separate regions of the VEGF-B cDNA or PDGF cDNA (oligonucleotides) are then added to the cDNA along with a polymerase such as Taq polymerase, and the polymerase amplifies the cDNA region between the two primers. [0166]
Preparation of a Vector for VEGF-B or PDGF Expression [0167]
After cloning, the cDNA or gene encoding a VEGF-B or PDGF polypeptide or fragment thereof has been isolated, it is preferably inserted into an amplification and/or expression vector in order to increase the copy number of the gene and/or to express the polypeptide in a suitable host cell and/or to transform cells in a target organism (to express VEGF-B or PDGF in vivo). Numerous commercially available vectors are suitable, though “custom made” vectors may be used as well. The vector is selected to be functional in a particular host cell or host tissue (i.e., the vector is compatible with the host cell machinery such that amplification of the VEGF-B or PDGF gene and/or expression of the gene can occur). The VEGF-B or PDGF polypeptide or fragment thereof may be amplified/expressed in prokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic host cells. Selection of the host cell will depend at least in part on whether the VEGF-B or PDGF polypeptide or fragment thereof is to be glycosylated. If so, yeast, insect, or mammalian host cells are preferable; yeast cells will glycosylate the polypeptide if a glycosylation site is present on the VEGF-B or PDGF amino acid sequence. [0168]
Typically, the vectors used in any of the host cells will contain 5′ flanking sequence and other regulatory elements as well such as an enhancer(s), an origin of replication element, a transcriptional termination element, a complete intron sequence containing a donor and acceptor splice site, a signal peptide sequence, a ribosome binding site element, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Optionally, the vector may contain a “tag” sequence, i.e., an oligonucleotide sequence located at the 5′ or 3′ end of the VEGF-B or PDGF coding sequence that encodes polyHis (such as hexaHis) or another small immunogenic sequence. This tag will be expressed along with the protein, and can serve as an affinity tag for purification of the VEGF-B or PDGF polypeptide from the host cell. Optionally, the tag can subsequently be removed from the purified VEGF-B or PDGF polypeptide by various means such as using a selected peptidase for example. [0169]
The vector/expression construct may optionally contain elements such as a 5′ flanking sequence, an origin of replication, a transcription termination sequence, a selectable marker sequence, a ribosome binding site, a signal sequence, and one or more intron sequences. The 5′ flanking sequence may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of p5′ flanking sequences from more than one source), synthetic, or it may be the native VEGF-B or [0170] PDGF 5′ flanking sequence. As such, the source of the 5′ flanking sequence may be any unicellular prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the 5′ flanking sequence is functional in, and can be activated by, the host cell machinery.
An origin of replication is typically a part of commercial prokaryotic expression vectors, and aids in the amplification of the vector in a host cell. Amplification of the vector to a certain copy number can, in some cases, be important for optimal expression of the VEGF-B or PDGF polypeptide. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector. [0171]
A transcription termination element is typically located 3′ to the end of the VEGF-B or PDGF polypeptide coding sequence and serves to terminate transcription of the VEGF-B or PDGF polypeptide. Usually, the transcription termination element in prokaryotic cells is a G-C rich fragment followed by a poly T sequence. Such elements can be cloned from a library, purchased commercially as part of a vector, and readily synthesized. [0172]
Selectable marker genes encode proteins necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells, (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media. [0173]
A ribosome binding element, commonly called the Shine-Dalgarno sequence (prokaryotes) or the Kozak sequence (eukaryotes), is necessary for translation initiation of mRNA. The element is typically located 3′ to the promoter and 5′ to the coding sequence of the polypeptide to be synthesized. The Shine-Dalgarno sequence is varied but is typically a polypurine (i.e., having a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using methods set forth above. [0174]
All of the elements set forth above, as well as others useful in this invention, are well known to the skilled artisan and are described, for example, in Sambrook, et al., “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Berger, et al., eds., “Guide To Molecular Cloning Techniques,” Academic Press, Inc., San Diego, Calif. (1987]. [0175]
For those embodiments of the invention where the recombinant VEGF-B or PDGF is to be secreted, a signal sequence is preferably included to direct secretion from the cell where it is synthesized. Typically, the signal sequence is positioned in the coding region of the transgene towards or at the 5′ end of the coding region. Many signal sequences have been identified, and any of them that are functional in the transgenic tissue may be used in conjunction with the transgene. Therefore, the signal sequence may be homologous or heterologous to the transgene, and may be homologous or heterologous to the transgenic mammal. Additionally, the signal sequence may be chemically synthesized using methods set forth above. However, for purposes herein, preferred signal sequences are those that occur naturally with the transgene (i.e., are homologous to the transgene). [0176]
In many cases, gene transcription is increased by the presence of one or more introns on the vector. The intron may be naturally-occurring within the transgene sequence, especially where the transgene is a full length or a fragment of a genomic DNA sequence. Where the intron is not naturally-occurring within the DNA sequence (as for most cDNAs), the intron(s) may be obtained from another source. The intron may be homologous or heterologous to the transgene and/or to the transgenic mammal. The position of the intron with respect to the promoter and the transgene is important, as the intron must be transcribed to be effective. As such, where the transgene is a cDNA sequence, the preferred position for the intron is 3′ to the transcription start site, and 5′ to the polyA transcription termination sequence. Preferably for cDNA transgenes, the intron will be located on one side or the other (i.e., 5′ or 3′) of the transgene sequence such that it does not interrupt the transgene sequence. Any intron from any source, including any viral, prokaryotic and eukaryotic (plant or animal) organisms, may be used to express VEGF-B or PDGF, provided that it is compatible with the host cell(s) into which it is inserted. Also included herein are synthetic introns. Optionally, more than one intron may be used in the vector. [0177]
Preferred vectors for recombinant expression of VEGF-B or PDGF protein are those that are compatible with bacterial, insect, and mammalian host cells. Such vectors include, inter alia, pCRII (Invitrogen Company, San Diego, Calif.), pBSII (Stratagene Company, La Jolla, Calif.), and pETL (BlueBacII; Invitrogen). [0178]
After the vector has been constructed and a VEGF-B or PDGF nucleic acid has been inserted into the proper site of the vector, the completed vector may be inserted into a suitable host cell for amplification and/or VEGF-B or PDGF polypeptide expression. The host cells typically used include, without limitation: Prokaryotic cells such as gram negative or gram positive cells, i.e., any strain of [0179] E. coli, Bacillus, Streptomyces, Saccharomyces, Salmonella, and the like; eukaryotic cells such as CHO (Chinese hamster ovary) cells, human kidney 293 cells, COS-7 cells; insect cells such as Sf4, Sf5, Sf9, and Sf21 and High 5 (all from the Invitrogen Company, San Diego, Calif.); and various yeast cells such as Saccharomyces and Pichia.
Insertion (also referred to as “transformation” or “transfection”) of the vector into the selected host cell may be accomplished using such methods as calcium chloride, electroporation, microinjection, lipofection or the DEAE-dextran method. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan, and are set forth, for example, in Sambrook, et al., supra. [0180]
The host cells containing the vector (i.e., transformed or transfected) may be cultured using standard media well known to the skilled artisan. The media will usually contain all nutrients necessary for the growth and survival of the cells. Suitable media for culturing [0181] E. coli cells are for example, Luria Broth (LB) and/or Terrific Broth (TB). Suitable media for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all of which may be supplemented with serum and/or growth factors as required by the particular cell line being cultured. A suitable medium for insect cultures is Grace's medium supplemented with yeastolate, lactalbumin hydrolysate, and/or fetal calf serum as necessary.
Typically, an antibiotic or other compound useful for selective growth of the transformed cells only is added as a supplement to the media. The compound to be used will be dictated by the selectable marker element present on the plasmid with which the host cell was transformed. For example, where the selectable marker element is kanamycin resistance, the compound added to the culture medium will be kanamycin. [0182]
The amount of VEGF-B or PDGF polypeptide produced in the host cell can be evaluated using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, HPLC separation, immunoprecipitation, and/or activity assays such as VEGFR-1, PDGFR-α, or PDGFR-β binding assays or cell stimulation assays. [0183]
Purification of VEGF-B or PDGF Polypeptides [0184]
VEGF-B polypeptides are preferably expressed and purified as described in U.S. Pat. No. 6,331,301, incorporated herein by reference. [0185]
If the VEGF-B or PDGF polypeptide has been designed to be secreted from the host cells, the majority of polypeptide will likely be found in the cell culture medium. If, however, the VEGF-B or PDGF polypeptide is not secreted from the host cells, it will be present in the cytoplasm (for eukaryotic, gram positive bacteria, and insect host cells) or in the periplasm (for gram negative bacteria host cells). [0186]
For intracellular VEGF-B or PDGF, the host cells are first disrupted mechanically or osmotically to release the cytoplasmic contents into a buffered solution. The polypeptide is then isolated from this solution. [0187]
Purification of VEGF-B or PDGF polypeptide from solution can be accomplished using a variety of techniques. If the polypeptide has been synthesized such that it contains a tag such as Hexahistidine (VEGF-B/hexaHis or PDGF/hexaHis) or other small peptide at either its carboxyl or amino terminus, it may essentially be purified in a one-step process by passing the solution through an affinity column where the column matrix has a high affinity for the tag or for the polypeptide directly (i.e., a monoclonal antibody specifically recognizing VEGF-B or PDGF). For example, polyhistidine binds with great affinity and specificity to nickel, thus an affinity column of nickel (such as the Qiagen nickel columns) can be used for purification of VEGF-B/polyHis or PDGF/polyHis. (See, for example, Ausubel, et al., eds., “Current Protocols In Molecular Biology,” Section 10.11.8, John Wiley & Sons, New York (1993)). [0188]
The strong affinity of VEGF-B for its receptor VEGFR-1 permits affinity purification of VEGF-B using an affinity matrix comprising VEGFR-1 extracellular domain. The strong affinity of PDGF-A for the PDGF receptor-α, the strong affinity for the PDGF-B for the PDGF receptors, the strong affinity of PDGF-C for the PDGF receptor-α and the strong affinity of PDGF-D receptors permit the affinity purification of these PDGFs using PDGF receptor-α and B extracellular domain. In addition, where the VEGF-B or PDGF polypeptide has no tag and no antibodies are available, other well known procedures for purification can be used. Such procedures include, without limitation, ion exchange chromatography, molecular sieve chromatography, HPLC, native gel electrophoresis in combination with gel elution, and preparative isoelectric focusing (“Isoprime” machine/technique, Hoefer Scientific). In some cases, two or more of these techniques may be combined to achieve increased purity. Preferred methods for purification include polyHistidine tagging and ion exchange chromatography in combination with preparative isoelectric focusing. [0189]
VEGF-B or PDGF polypeptide found in the periplasmic space of the bacteria or the cytoplasm of eukaryotic cells, the contents of the periplasm or cytoplasm, including inclusion bodies (bacteria) if the processed polypeptide has formed such complexes, can be extracted from the host cell using any standard technique known to the skilled artisan. For example, the host cells can be lysed to release the contents of the periplasm by French press, homogenization, and/or sonication. The homogenate can then be centrifuged. [0190]
If the VEGF-B or PDGF polypeptide has formed inclusion bodies in the periplasm, the inclusion bodies can often bind to the inner and/or outer cellular membranes and thus will be found primarily in the pellet material after centrifugation. The pellet material can then be treated with a chaotropic agent such as guanidine or urea to release, break apart, and solubilize the inclusion bodies. The VEGF-B or PDGF polypeptide in its now soluble form can then be analyzed using gel electrophoresis, immunoprecipitation or the like. If it is desired to isolate the VEGF-B or PDGF polypeptide, isolation may be accomplished using standard methods such as those set forth below and in Marston, et al., [0191] Meth. Enz., 182:264-275 (1990).
If VEGF-B or PDGF polypeptide inclusion bodies are not formed to a significant degree in the periplasm of the host cell, the VEGF-B or PDGF polypeptide will be found primarily in the supernatant after centrifugation of the cell homogenate, and the VEGF-B or PDGF polypeptide can be isolated from the supernatant using methods such as those set forth below. [0192]
In those situations where it is preferable to partially or completely isolate the VEGF-B or PDGF polypeptide, purification can be accomplished using standard methods well known to the skilled artisan. Such methods include, without limitation, separation by electrophoresis followed by electroelution, various types of chromatography (immunoaffinity, molecular sieve, and/or ion exchange), and/or high pressure liquid chromatography. In some cases, it may be preferable to use more than one of these methods for complete purification. [0193]
Anti-VEGF-B or Anti-PDGF Therapeutic Compounds [0194]
Anti-VEGF-B or Anit-PDGF therapies as discussed below include, but are not limited to antibody, aptamer, antisense and interference RNA techniques and therapies. These therapies are directed to myelosuppression instead of myelopoiesis. Whereas myelosuppression is often what one seeks to treat, for some conditions and disease states, such as in leukemia and lymphoma, myelosuppression may be desirable. [0195]
Therapeutic Anti-VEGF-B or Anti-PDGF Antibodies [0196]
Anti-VEGF-B antibodies as described in U.S. Pat. No. 6,331,301 are also contemplated for use in practicing the present invention. Such antibodies can be used for VEGF-B purification as described above, or therapeutically where inhibition of VEGF-B is desired (e.g., to achieve myelosuppressive effects). [0197]
Polyclonal or monoclonal therapeutic anti-VEGF-B or Anti-PDGF antibodies useful in practicing this invention may be prepared in laboratory animals or by recombinant DNA techniques using the following methods. Polyclonal antibodies to the VEGF-B or PDGF molecule or a fragment thereof containing the target amino acid sequence generally are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the VEGF-B or PDGF molecule in combination with an adjuvant such as Freund's adjuvant (complete or incomplete). To enhance immunogenicity, it may be useful to first conjugate the VEGF-B or PDGF molecule or a fragment containing the target amino acid sequence of to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R.sup. 1 N═C=NR, where R and R[0198] ¹are different alkyl groups. Alternatively, VEGF-B-immunogenic conjugates can be produced recombinantly as fusion proteins.
Animals are immunized against the immunogenic VEGF-B or PDGF conjugates or derivatives (such as a fragment containing the target amino acid sequence) by combining about 1 mg or about 1 microgram of conjugate (for rabbits or mice, respectively) with about 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. Approximately 7 to 14 days later, animals are bled and the serum is assayed for anti-VEGF-B or PDGF titer. Animals are boosted with antigen repeatedly until the titer plateaus. Preferably, the animal is boosted with the same VEGF-B or PDGF molecule or fragment thereof as was used for the initial immunization, but conjugated to a different protein and/or through a different cross-linking agent. In addition, aggregating agents such as alum are used in the injections to enhance the immune response. [0199]
Monoclonal antibodies may be prepared by recovering spleen cells from immunized animals and immortalizing the cells in conventional fashion, e.g. by fusion with myeloma cells. The clones are then screened for those expressing the desired antibody. The monoclonal antibody preferably does not cross-react with other VEGF or PDGF family members. [0200]
Preparation of antibodies using recombinant DNA methods such as the phagemid display method, may be accomplished using commercially available kits, as for example, the Recombinant Phagemid Antibody System available from Pharmacia (Uppsala, Sweden), or the SurfZAP™ phage display system (Stratagene Inc., La Jolla, Calif.). [0201]
Preferably, antibodies for administration to humans, although prepared in a laboratory animal such as a mouse, will be “humanized”, or chimeric, i.e. made to be compatible with the human immune system such that a human patient will not develop an immune response to the antibody. Even more preferably, human antibodies which can now be prepared using methods such as those described for example, in Lonberg, et al., [0202] Nature Genetics, 7:13-21 (1994) are preferred for therapeutic administration to patients.
A. Humanization of Anti-VEGF-B or Anti-PDGF Monoclonal Antibodies [0203]
VEGF-B-neutralizing antibodies comprise one class of therapeutics useful as VEGF-B antagonists. PDGF-neutralizing antibodies comprise one class of therapeutics useful as PDGF antagonists. Following are protocols to improve the utility of anti-VEGF-B monoclonal antibodies as therapeutics in humans, by “humanizing” the monoclonal antibodies to improve their serum half-life and render them less immunogenic in human hosts (i.e., to prevent human antibody response to non-human anti-VEGF-B or non-human anti-PDGF antibodies). [0204]
The principles of humanization have been described in the literature and are facilitated by the modular arrangement of antibody proteins. To minimize the possibility of binding complement, a humanized antibody of the IgG4 isotype is preferred. [0205]
For example, a level of humanization is achieved by generating chimeric antibodies comprising the variable domains of non-human antibody proteins of interest, such as the anti-VEGF-B monoclonal antibodies described herein, with the constant domains of human antibody molecules. (See, e.g., Morrison and Oi, [0206] Adv. Immunol., 44:65-92 (1989)). The variable domains of VEGF-B neutralizing anti-VEGF-B antibodies are cloned from the genomic DNA of a B-cell hybridoma or from cDNA generated from mRNA isolated from the hybridoma of interest. The V region gene fragments are linked to exons encoding human antibody constant domains, and the resultant construct is expressed in suitable mammalian host cells (e.g., myeloma or CHO cells).
To achieve an even greater level of humanization, only those portions of the variable region gene fragments that encode antigen-binding complementarity determining regions (“CDR”) of the non-human monoclonal antibody genes are cloned into human antibody sequences. (See, e.g., Jones, et al., [0207] Nature, 321:522-525 (1986); Riechmann, et al., Nature, 332:323-327 (1988); Verhoeyen, et al., Science, 239:1534-36 (1988); and Tempest, et al., Bio/Technology, 9:266-71 (1991)). If necessary, the beta-sheet framework of the human antibody surrounding the CDR3 regions also is modified to more closely mirror the three dimensional structure of the antigen-binding domain of the original monoclonal antibody. (See, Kettleborough, et al., Protein Engin., 4:773-783 (1991); and Foote, et al., J. Mol. Biol., 224:487-499 (1992)).
In an alternative approach, the surface of a non-human monoclonal antibody of interest is humanized by altering selected surface residues of the non-human antibody, e.g., by site-directed mutagenesis, while retaining all of the interior and contacting residues of the non-human antibody. See Padlan, [0208] Molecular Immunol., 28(4/5):489-98 (1991).
The foregoing approaches are employed using VEGF-B-neutralizing anti-VEGF-B monoclonal antibodies and the hybridomas that produce them to generate humanized VEGF-B-neutralizing antibodies useful as therapeutics to treat or palliate conditions wherein VEGF-B expression is detrimental. [0209]
The foregoing approaches are employed using PDGF-neutralizing anti-PDGF monoclonal antibodies and the hybridomas that produce them to generate humanized PDGF-neutralizing antibodies useful as therapeutics to treat or palliate conditions wherein PDGF expression is detrimental. [0210]
B. Human VEGF-B-Neutralizing or Human PDGF-Neutralizing Antibodies from Phage Display [0211]
Human VEGF-B-neutralizing or PDGF-neutralizing antibodies are generated by phage display techniques such as those described in Aujame, et al., [0212] Human Antibodies, 8(4):155-168 (1997); Hoogenboom, TIBTECH, 15:62-70 (1997); and Rader, et al., Curr. Opin. Biotechnol., 8:503-508 (1997), all of which are incorporated by reference. For example, antibody variable regions in the form of Fab fragments or linked single chain Fv fragments are fused to the amino terminus of filamentous phage minor coat protein pIII. Expression of the fusion protein and incorporation thereof into the mature phage coat results in phage particles that present an antibody on their surface and contain the genetic material encoding the antibody. A phage library comprising such constructs is expressed in bacteria, and the library is panned (screened) for VEGF-B-specific or PDGF-specific phage-antibodies using labeled or immobilized VEGF-B or PDGF respectively as antigen-probe.
C. Human VEGF-B-Neutralizing or Human PDGF-Neutralizing Antibodies from Transgenic Mice [0213]
Human VEGF-B-neutralizing antibodies are generated in transgenic mice essentially as described in Bruggemann and Neuberger, [0214] Immunol. Today, 17(8):391-97 (1996) and Bruggemann and Taussig, Curr. Opin. Biotechnol., 8:455-58 (1997). Transgenic mice carrying human V-gene segments in germline configuration and that express these transgenes in their lymphoid tissue are immunized with VEGF-B or PDGF composition using conventional immunization protocols. Hybridomas are generated using B cells from the immunized mice using conventional protocols and screened to identify hybridomas secreting anti-VEGF-B or anti-PDGF human antibodies (e.g., as described above).
D. Bispecific Antibodies [0215]
Bispecific antibodies that specifically bind to one protein (e.g., VEGF-B or PDGF) and that specifically bind to other antigens relevant to pathology and/or treatment are produced, isolated, and tested using standard procedures that have been described in the literature. See, e.g., Pluckthun & Pack, [0216] Immunotechnology, 3:83-105 (1997); Carter, et al., J. Hematotherapy, 4: 463-470 (1995); Renner & Pfreundschuh, Immunological Reviews, 1995, No. 145, pp. 179-209; Pfreundschuh U.S. Pat. No. 5,643,759; Segal, et al., J. Hematotherapy, 4: 377-382 (1995); Segal, et al., Immunobiology, 185: 390-402 (1992); and Bolhuis, et al., Cancer Immunol. Immunother., 34: 1-8 (1991), all of which are incorporated herein by reference in their entireties.
Anti-VEGF-B and Anti-PDGF Aptamers [0217]
Recent advances in the field of combinatorial sciences have identified short polymer sequences with high affinity and specificity to a given target. For example, SELEX technology has been used to identify DNA and RNA aptamers with binding properties that rival mammalian antibodies, the field of immunology has generated and isolated antibodies or antibody fragments which bind to a myriad of compounds and phage display has been utilized to discover new peptide sequences with very favorable binding properties. Based on the success of these molecular evolution techniques, it is certain that ligands can be created which bind to any molecule. Curiously, in each case, a loop structure is often involved with providing the desired binding attributes as in the case of: aptamers which often utilize hairpin loops created from short regions without complimentary base pairing, naturally derived antibodies that utilize combinatorial arrangement of looped hyper-variable regions and new phage display libraries utilizing cyclic peptides that have shown improved results when compare to linear peptide phage display results. Thus, sufficient evidence has been generated to suggest that high affinity ligands can be created and identified by combinatorial molecular evolution techniques. For the present invention, molecular evolution techniques can be used to isolate ligands specific for VEGF-B, to be used in a manner analogous to that discussed above for anti-VEGF-B antibodies. For more on aptamers, see generally, Gold, L., Singer, B., He, Y. Y., Brody. E., “Aptamers As Therapeutic And Diagnostic Agents,” [0218] J. Biotechnol. 74:5-13 (2000).
Anti-sense Molecules and Therapy [0219]
Another class of VEGF-B or PDGF inhibitors useful in the present invention is isolated antisense nucleic acid molecules that can hybridize to, or are complementary to, the nucleic acid molecule comprising the VEGF-B or PDGF nucleotide sequence, or fragments, analogs or derivatives thereof. An “antisense” nucleic acid comprises a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire VEGF-B or PDGF coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of VEGF-B or PDGF or antisense nucleic acids complementary to a VEGF-B or PDGF nucleic acid sequence are additionally provided. [0220]
In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding a VEGF-B or PDGF protein. The term “coding region” refers to the region of the nucleotide sequence comprising codons that are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a “conceding region” of the coding strand of a nucleotide sequence encoding the VEGF-B or PDGF protein. The term “conceding region” refers to 5′ and 3′ sequences that flank the coding region and that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions). [0221]
Given the coding strand sequences encoding the VEGF-B or PDGF protein disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of VEGF-B or PDGF mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of VEGF-B or PDGF mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of VEGF-B mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used). [0222]
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following section). [0223]
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding VEGF-B or PDGF to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred. [0224]
In yet another embodiment, the antisense nucleic acid molecule of the invention is an alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual alpha-units, the strands run parallel to each other. See, e.g., Gaultier, et al., [0225] Nucl. Acids Res., 15:6625-6641 (1987). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (see, e.g., Inoue, et al. Nucl. Acids Res., 15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (see, e.g., Inoue, et al., FEBS Lett., 215:327-330 (1987)).
Production and delivery of antisense molecules are facilitated by providing a vector comprising an anti-sense nucleotide sequence complementary to at least a part of the VEGF-B or PDGF DNA sequence. According to a yet further aspect of the invention such a vector comprising an anti-sense sequence may be used to inhibit, or at least mitigate, VEGF-B or PDGF expression. The use of a vector of this type to inhibit VEGF-B or PDGF expression is favored in instances where VEGF-B or PDGF expression is associated with a particular disease state. [0226]
Anti-VEGF-B or Anit-PDGF RNA Interference [0227]
Use of RNA Interference to inactivate or modulate VEGF-B or PDGF expression is also contemplated by this invention. RNA interference is described in U.S. Patent Appl. No. 2002-0162126, and Hannon, G., [0228] J. Nature, 11:418:244-51 (2002). “RNA interference,” “post-transcriptional gene silencing,” “quelling”—these terms have all been used to describe similar effects that result from the overexpression or misexpression of transgenes, or from the deliberate introduction of double-stranded RNA into cells (reviewed in Fire, A., Trends Genet 15:358-363 (1999); Sharp, P. A., Genes Dev., 13:139-141 (1999); Hunter, C., Curr. Biol., 9:R440-R442 (1999); Baulcombe, D. C., Curr. Biol. 9:R599-R601 (1999); Vaucheret, et al. Plant J. 16:651-659 (1998), all incorporated by reference. RNA interference, commonly referred to as RNAi, offers a way of specifically and potently inactivating a cloned gene.
Therapeutic Compositions and Administration [0229]
Therapeutic formulations of the compositions useful for practicing the present invention such as VEGF-B polypeptides, polynucleotides, or antibodies may be prepared for storage by mixing the selected composition having the desired degree of purity with optional physiologically pharmaceutically-acceptable carriers, excipients, or stabilizers (Remington's [0230] Pharmaceutical Sciences, 18th edition, A. R. Gennaro, ed., Mack Publishing Company (1990)) in the form of a lyophilized cake or an aqueous solution. Acceptable carriers, excipients or stabilizers are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG).
The composition to be used for in vivo administration should be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution. The composition for parenteral administration ordinarily will be stored in lyophilized form or in solution. [0231]
Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The route of administration of the composition is in accord with known methods, e.g. oral, injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional routes, or by sustained release systems or implantation device. Where desired, the compositions may be administered continuously by infusion, bolus injection or by implantation device. [0232]
Suitable examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman, et al., [0233] Biopolymers, 22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer, et al., J. Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer, et al., supra) or poly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained-release compositions also may include liposomes, which can be prepared by any of several methods known in the art (e.g., DE 3,218,121; Epstein, et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA, 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949).
An effective amount of the compositions to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage may range from about 1 μg/kg to up to 100 mg/kg or more, depending on the factors mentioned above. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays designed to evaluate myelosuppression or the particular conditions of interest in a particular subject. [0234]
Pharmaceutical compositions may be produced by admixing a pharmaceutically effective amount of VEGF-B protein with one or more suitable carriers or adjuvants such as water, mineral oil, polyethylene glycol, starch, talcum, lactose, thickeners, stabilizers, suspending agents, etc. Such compositions may be in the form of solutions, suspensions, tablets, capsules, creams, salves, ointments, or other conventional forms. [0235]
VEGF-B or PDGFs can be used directly to practice materials and methods of the invention, but in preferred embodiments, the compounds are formulated with pharmaceutically acceptable diluents, adjuvants, excipients, or carriers. The phrase “pharmaceutically or pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human, e.g., orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. (The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intracistemal injection, or infusion techniques. Administration by intravenous, intradermal, intramusclar, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a particular site is contemplated as well.) Generally, this will also entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. The term “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. [0236]
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. [0237]
Polynucleotide-Based VEGF-B or PDGF Therapies [0238]
In one embodiment, the therapeutic effects of VEGF-B or PDGFs on stem cell recruitment, proliferation, and/or differentiation are achieved by administration of VEGF-B or PDGF encoding polynucleotides (including vectors comprising such polynucleotides) to a subject that will benefit from the VEGF-B or PDGF. [0239]
For these embodiments, an exemplary expression construct comprises a virus or engineered construct derived from a viral genome. The expression construct generally comprises a nucleic acid encoding the gene to be expressed and also additional regulatory regions that will effect the expression of the gene in the cell to which it is administered. Such regulatory regions include for example promoters, enhancers, polyadenylation signals and the like. [0240]
It is now widely recognized that DNA may be introduced into a cell using a variety of viral vectors. In such embodiments, expression constructs comprising viral vectors containing the genes of interest may be adenoviral (see, for example, U.S. Pat. No. 5,824,544; U.S. Pat. No. 5,707,618; U.S. Pat. No. 5,693,509; U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,585,362; each incorporated herein by reference), retroviral (see, for example, U.S. Pat. No. 5,888,502; U.S. Pat. No. 5,830,725; U.S. Pat. No. 5,770,414; U.S. Pat. No. 5,686,278; U.S. Pat. No. 4,861,719 each incorporated herein by reference), adeno-associated viral (see, for example, U.S. Pat. No. 5,474,935; U.S. Pat. No. 5,139,941; U.S. Pat. No. 5,622,856; U.S. Pat. No. 5,658,776; U.S. Pat. No. 5,773,289; U.S. Pat. No. 5,789,390; U.S. Pat. No. 5,834,441; U.S. Pat. No. 5,863,541; U.S. Pat. No. 5,851,521; U.S. Pat. No. 5,252,479 each incorporated herein by reference), an adenoviral-adenoassociated viral hybrid (see, for example, U.S. Pat. No. 5,856,152 incorporated herein by reference) or a vaccinia viral or a herpesviral (see, for example, U.S. Pat. No. 5,879,934; U.S. Pat. No. 5,849,571; U.S. Pat. No. 5,830,727; U.S. Pat. No. 5,661,033; U.S. Pat. No. 5,328,688 each incorporated herein by reference) vector. [0241]
In other embodiments, non-viral delivery is contemplated. These include calcium phosphate precipitation (Graham and Van Der Eb, [0242] Virology, 52:456-467 (1973); Chen and Okayama, Mol. Cell Biol., 7:2745-2752, (1987); Rippe, et al., Mol. Cell Biol., 10:689-695 (1990)), DEAE-dextran (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), electroporation (Tur-Kaspa, et al., Mol. Cell Biol., 6:716-718, (1986); Potter, et al., Proc. Nat. Acad. Sci. USA, 81:7161-7165, (1984)), direct microinjection (Harland and Weintraub, J. Cell Biol., 101: 1094-1099 (1985)), DNA-loaded liposomes (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982); Fraley, et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352 (1979); Felgner, Sci. Am., 276(6):102-6 (1997); Felgner, Hum. Gene Ther., 7(15):1791-3, (1996)), cell sonication (Fechheimer, et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467 (1987)), gene bombardment using high velocity microprojectiles (Yang, et al., Proc. Natl. Acad. Sci. USA, 87:9568-9572 (1990)), and receptor-mediated transfection (Wu and Wu, J. Biol. Chem., 262:4429-4432 (1987); Wu and Wu, Biochemistry, 27:887-892 (1988); Wu and Wu, Adv. Drug Delivery Rev., 12:159-167 (1993)).
In a particular embodiment of the invention, the expression construct (or indeed the peptides discussed above) may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, “In Liver Diseases, Targeted Diagnosis And Therapy Using Specific Receptors And Ligands,” Wu, G., Wu, C., ed., New York: Marcel Dekker, pp. 87-104 (1991)). The addition of DNA to cationic liposomes causes a topological transition from liposomes to optically birefringent liquid-crystalline condensed globules (Radler, et al., [0243] Science, 275(5301):810-4, (1997)). These DNA-lipid complexes are potential non-viral vectors for use in gene therapy and delivery.
Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Also contemplated in the present invention are various commercial approaches involving “lipofection” technology. In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda, et al., [0244] Science, 243:375-378 (1989)). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato, et al., J. Biol. Chem., 266:3361-3364 (1991)). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.
Other vector delivery systems that can be employed to deliver a nucleic acid encoding a therapeutic gene into cells include receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu (1993), supra). [0245]
Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu (1987), supra) and transferrin (Wagner, et al., [0246] Proc. Nat'l. Acad. Sci. USA, 87(9):3410-3414 (1990)). Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol, et al., FASEB. J., 7:1081-1091 (1993); Perales, et al., Proc. Natl. Acad. Sci., USA 91:4086-4090 (1994)) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).
In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau, et al., [0247] Methods Enzymol., 149:157-176 (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a therapeutic gene also may be specifically delivered into a particular cell type by any number of receptor-ligand systems with or without liposomes.
In another embodiment of the invention, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above that physically or chemically permeabilize the cell membrane. This is applicable particularly for transfer in vitro, however, it may be applied for in vivo use as well. Dubensky, et al., [0248] Proc. Nat. Acad. Sci. USA, 81:7529-7533 (1984) successfully injected polyomavirus DNA in the form of CaPO₄precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif, Proc. Nat. Acad. Sci. USA, 83:9551-9555 (1986) also demonstrated that direct intraperitoneal injection of CaPO₄precipitated plasmids results in expression of the transfected genes.
Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein, et al., [0249] Nature, 327:70-73 (1987)). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang, et al., Proc. Natl. Acad. Sci USA, 87:9568-9572 (1990)). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
Those of skill in the art are well aware of how to apply gene delivery to in vivo and ex vivo situations. For viral vectors, one generally will prepare a viral vector stock. Depending on the kind of virus and the titer attainable, one will deliver 1×10[0250] ⁴, 1×10⁵, 1×10×⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹or 1×10¹²infectious particles to the patient. Similar figures may be extrapolated for liposomal or other non-viral formulations by comparing relative uptake efficiencies. Formulation as a pharmaceutically acceptable composition is discussed below.
Various routes are contemplated for various cell types. For practically any cell, tissue or organ type, systemic delivery is contemplated. In other embodiments, a variety of direct, local and regional approaches may be taken. For example, the cell, tissue or organ may be directly injected with the expression vector or protein. [0251]
Preferred promoters for gene therapy for use in this invention include cytomegalovirus (CMV) promoter/enhancer, long terminal repeat (LTR) of retroviruses, keratin 14 promoter, and a myosin heavy chain promoter. [0252]
In a different embodiment, ex vivo gene therapy is contemplated. In an ex vivo embodiment, cells from the patient are removed and maintained outside the body for at least some period of time. During this period, a therapy is delivered, after which the cells are reintroduced into the patient; preferably, any tumor cells in the sample have been killed. [0253]
The techniques, procedures and methods outlined herein for VEGF-B and PDGF are applicable to any and all of the growth factors of the present invention. [0254]
The invention may be more readily understood by reference to the following examples, are given to illustrate the invention and not in any way to limit its scope. [0255]

EXAMPLE 1

Effects of VEGF-B or VEGF-C Gene Therapy on White Blood Cell Counts

The following procedures were performed to elucidate the roles of certain growth factors and their receptors, including VEGF-B and its receptor VEGFR-1, on hematopoietic progenitor cells. [0256]
NMRI nu/nu mice (nude mice) received intravenous injection of adenoviruses encoding one of the following proteins: beta-galactosidase (1×10[0257] ⁹pfu), VEGF-C (3×10⁸pfu), VEGF-C 156S (a mutant form of VEGF-C; 3×10⁸pfu, see U.S. Pat. Number 6,361,946), a soluble form of the VEGFR-3 extracellular domain (VEGFR-3-Ig fusion protein; 1×10⁹pfu), or VEGF-B (50:50 mixture of VEGF-B₁₆₇and VEGF-B₁₈₆) (1×10⁹pfu). The beta-galactosidase served as a negative control.
Four days after the injection the mice were sacrificed, blood was collected and white blood cell (WBC) counts from the peripheral blood were measured using flow cytometry. In addition, WBCs of the blood were treated with anti-VEGFR-3 antibodies and stained with phycoerythrin-conjugated rat anti-mouse IgG2A for fluorescence activated cell sorting (FACS) analysis. Red blood cells were lysed by a buffered ammonium chloride/potassium (ACK) lysing solution and 1×10[0258] ⁵white blood cells were used per sample.
The white blood cells were washed with PBS containing 2% fetal calf serum and incubated with Fc Block (BD Pharmingen) for 5 minutes followed by incubation with conjugated antibody for 30 minutes on ice. As a negative isotype control, phycoerythrin-conjugated rat IgG2A (BD Pharmingen) was used at the same concentration to measure the background signal. Cells were washed and analyzed in the LSR cytometer (Becton Dickinson). Other PE- or FITC-labeled antibodies used in the flow cytometry were anti-VEGFR-1, anti-Tie-2, anti-VEGFR-2, anti-CD34, anti-CD117, anti-CD11b, and anti-Ly-6G/C. [0259]
In mice treated with Ad-VEGF-C156S, a clear mobilization of hematopoietic cells expressing VEGFR-1, VEGFR-2 and VEGFR-3 to the peripheral blood was seen. Furthermore, the percentage of CD34+cells in the blood circulation was higher when compared to the Ad-LacZ treated mice. [0260]
Elevated numbers of VEGFR-1[0261] ⁺ cells were also present in the Ad-VEGF-C and Ad-VEGF-B groups (FIG. 3a-d). The results indicate that VEGF-C, and especially its VEGFR-3 specific mutant form VEGF-C156S, as well as VEGF-B can mobilize endothelial/hematopoietic progenitor cells from the bone marrow.

EXAMPLE 2

Recovery from Chemotherapy-Induced Myelosuppression

To study hematopoiesis during bone marrow recovery, FVB or NMRI wild-type mice (6-10 weeks old) were treated with a single i.v. injection of cytotoxic 5-fluorouracil (5-FU, 300 mg/kg, Pharmacia), which transiently depletes most of the circulating hematopoietic cells. Recovery with and without various exogenous growth factor treatments was studied. WBCs were analyzed as described in Example 1. [0262]
In a first experiment, the effects of 5-FU alone were studied. Before the myelosuppressive treatment, the peripheral blood contained about 4.1% white blood cells (WBCs) positive for VEGFR-3 staining. The percentage of VEGFR-3+cells in the blood increased beginning on [0263] day 5 after 5-FU treatment, and on day 16 the number was 23.7% (FIG. 1a). Also, in the bone marrow of the femur the percentage of VEGFR-3 positive cells was clearly elevated after 5-FU treatment (FIG. 1b).
In a second set of experiments, mice treated with 5-FU simultaneously received an intravenous injection of an adenoviruses encoding one of the following proteins: beta-galactosidase (1×10[0264] ⁹pfu), VEGF-C (3×10⁸pfu), VEGF-C156S (3×10⁸pfu), or soluble VEGFR-3 extracellular domain (VEGFR-3-1 g fusion protein; 1×10⁹pfu).
White blood counts (WBC) from the peripheral blood were measured after two or four days using the techniques described in Example 1. In Ad-VEGF-C treated mice, the number of WBC was higher during the first four days (+43% at day 4), whereas in AdVEGFR-3-Ig treated mice WBC count decreased more rapidly (−27% at [0265] day 2, −12% at day 4), when compared to Ad-LacZ treated mice (FIG. 2a). Furthermore, the injection of adenoviruses encoding VEGF-C156S, a mutant form of VEGF-C, which activates only VEGFR-3, also increased the WBC number in peripheral blood in the same 5-FU model (FIG. 2b).
The mice received second injections of adenoviruses encoding VEGF-C or VEGFR-3-Ig on [0266] day 10. In VEGFR-3-Ig treated mice, blocking the VEGFR-3 pathway inhibited the bone marrow recovery and elevation of the WBC number (FIG. 2c).

EXAMPLE 3

Effects of VEGF-B or PDGF Gene Therapy on White Blood Cell Counts

The following procedures are performed to elucidate the roles of certain growth factors and their receptors, including VEGF-B and its receptor VEGFR-1 and the PDGFs and their respective receptors, on hematopoietic progenitor cells. [0267]
NMRI nu/nu mice (nude mice), VEGF-B deficient mice (VEGF-B knock-out mice as described in Aase, et al., [0268] Circulation, 104:358-64 (2001) and Wanstall, et al., Card. Res., 55:361-368 (2002)), or PDGF (PDGF-A, PDGF-B, PDGF-C, or PDGF-D) deficient mice receive intravenous injection of adenoviruses encoding one or more of the following proteins at concentrations of 8×10⁷to 6×10⁹pfu: beta-galactosidase, VEGF-B₁₆₇, VEGF-B₁₈₆, a VEGF-B N-acetylated variant, PDGF-A, PDGF-B, PDGF-C, and PDGF-D. The beta-galactosidase serves as a negative control.
Four days after the viral injection, the mice are sacrificed, blood is collected and white blood cell (WBC) counts from the peripheral blood were measured after four days using flow cytometry. Red blood cells are lysed by a buffered ammonium chloride/potassium (ACK) lysing solution and 10[0269] ⁵white blood cells are used per sample. White blood cells are immunoanalyzed as in Example 1, and additionally with anti-PDGF-receptor-α and anti-PDGF-receptor-β antibodies.
The relative number of white blood cells expressing the various cell-surface markers indicative of stem cells are compared between control and experimental mice to evaluate the level of stem cell recruitment, differentiation and proliferation. [0270]

EXAMPLE 4

Myelosuppression and Recovery with VEGF-B and PDGFS

To study hematopoiesis during bone marrow recovery, FVB or NMRI wild-type mice (6-10 weeks old), VEGF-B-deficient or PDGF (PDGF-A, PDGF-B, PDGF-C, or PDGF-D) deficient mice are treated with a single i.v. injection of cytotoxic 5-fluorouracil (5-FU, 300 mg/kg, Pharmacia), which transiently depletes most of the circulating hematopoietic cells. Recovery with and without various exogenous growth factor treatments is studied. WBCs are analyzed as described in Example 1, and additionally with anti-PDGF-receptor-α and anti-PDGF-receptor-β antibodies. [0271]
In a first experiment, the effects of 5-FU alone are studied. Before the myelosuppressive treatment, the peripheral blood is analyzed according to Example 2, and additionally with anti-PDGF-receptor-α and anti-PDGF-receptor-β antibodies. [0272]
In a second set of experiments, mice are treated simultaneously with 5-FU and with adenoviruses (at concentrations of 8×10[0273] ⁷to 6×10⁹pfu) containing transgenes encoding one or more of the following proteins: beta-galactosidase, VEGF-B₁₆₇, VEGF-B₁₈₆, a VEGF-B N-acetylated variant, PDGF-A, PDGF-B, PDGF-C, and PDGF-D. In addition, adenoviruses encoding solubilized PDGF receptor extracellular domain/IgG Fusion are tested.
White blood counts (WBC) from the peripheral blood are analyzed after two or four days using the techniques described in Example 2, and additionally with anti-PDGF-receptor-α and anti-PDGF-receptor-β antibodies. [0274]
The relative number of white blood cells expressing the various cell-surface markers indicative of stem cells (e.g., AC133, VEGFR-2 or -1, c-kit) are compared between control and experimental mice to evaluate the effects of each protein on stem cell recruitment, differentiation and proliferation. [0275]

EXAMPLE 5

Myelopoietic Protein Therapy

The following procedures are performed to elucidate the roles of certain growth factors and their receptors, including VEGF-B and its receptor VEGFR-1 and the PDGFs and their respective receptors, on hematopoietic progenitor cells. [0276]
NMRI nu/nu mice (nude mice), VEGF-B deficient mice (VEGF-B knock-out mice as described in Aase, et al., [0277] Circulation, 104:358-64 (2001) and Wanstall, et al., Card. Res., 55:361-368 (2002)), or PDGF (PDGF-A, PDGF-B, PDGF-C, or PDGF-D) deficient mice receive a control protein or one or more of the following growth factors: VEGF-B₁₆₇, VEGF-B₁₈₆, a VEGF-B N-acetylated variant, PDGF-A, PDGF-B, PDGF-C, and PDGF-D. Alternatively, the mice receive soluble receptor extracellular domain protein preparations (e.g., VEGFR-1-Ig, PDGFR-α-Ig, or PDGFR-β-Ig). Before administering the protein compositions, baseline white blood cells are characterized as described in Example 1. The mice receive an initial intravenous (IV) bolus dose of growth factor or control protein over 60 minutes. After a 48-hour observation period, the mice receive a 14-day course of continuous IV infusion of the growth factor or control protein. A variety of protein concentrations are tested. For example, the mice receive a total dose of either 0.5 1.0, 2.0, 4.0, or 8.0 μg/kg. On days 2, 6, 11, 16 and 24, white blood cells are characterized as described in Example 1.
The relative number of white blood cells expressing the various cell-surface markers indicative of stem cells are compared between control and experimental mice to evaluate the effects of each protein on stem cell recruitment, differentiation and proliferation. [0278]

EXAMPLE 6

Ex Vivo Expansion of Endothelial Progenitor Cells Derived from Subjects Treated with VEGF-B or PDGFS

The following experiments are performed to demonstrate the ability of VEGF-B and/or PDGF therapy to improve the efficacy and healing of a tissue, organ, or prosthetic graft or implant. This example is based on the methods of Kaushal, et al., “Functional Small-Diameter Neovessels Created Using Endothelial Progenitor Cells Expanded Ex Vivo,” [0279] Nat. Med., 7:1035-1040 (2001), which is incorporated herein in its entirety. A subject is treated with one or more of the following: VEGF-B₁₆₇, VEGF-B₁₈₆, a VEGF-B N-acetylated variant, PDGF-A, PDGF-B, PDGF-C, and PDGF-D or a control either in direct protein form or encoded by a polynucleotide as part of a gene therapy vector, such as a recombinant adenovirus, adeno-associated virus (AAV), plasmid or other vector, or naked DNA comprising a polynucleotide that encodes VEGF-B, a PDGF, or a fragment thereof. In one variation, VEGF-B and/or a PDGF protein is administered using implantable osmotic mini-pumps. The VEGF-B or PDGF therapy is performed to increase the quantity of circulating endothelial progenitor cells (EPCs).
After 2, 4, 6, 8, 12 or 14 days of treatment as described above, blood is drawn in heparinized tubes and the leukocytes are isolated on a Histopaque density gradient (Sigma) for 30 minutes at 1000 g using Accuspin tubes (Sigma). The leukocytes are resuspended in growth medium (e.g. EBM-2 medium (Clonetics, San Diego)) with 20% fetal calf serum and plated on fibronectin coated plates. Adhering cells are then expanded (preferably in the presence of a VEGF-B or a PDGF, 1-10 ug/ml of growth medium). Preferably, a sample from the cells is analyzed for cell surface molecules indicative of undifferentiated and differentiating progenitor cells. [0280]
Subjects are divided into two groups for mock surgery or surgery to implant or transplant a prosthesis or tissue or organ graft, such as a skin, bone, ligament, tendon, cartilage, vein or arterial graft. See Tepper, et al., “Endothelial Progenitor Cells: The Promise Of Vascular Stem Cells For Plastic Surgery,” [0281] Plastic and Reconstructive Surgery, 111:846-854 (2003). For the experimental group, the expanded progenitor cells are isolated from the plates and seeded into the surgical wounds, transplants or grafts (including synthetic grafts employing tissue engineering), or are reintroduced intravenously into the circulating blood. Control animals receive no cell therapy or cell therapy using nucleated cells isolated as described above, but without growth factor pretreatment and without growth factor-supplemented culture.
Animals are examined or sacrificed at various time points to evaluate the speed with which the wounds have healed and/or the success with which the body has accepted the graft or transplant. [0282]

EXAMPLE 7

VEGF-B, Chemotherapy, Bone Marrow Transplant Study

Four groups of test animals are established, one group will receive neither chemotherapy nor a bone marrow transplantation, one group will receive chemotherapy using one or more of the chemotherapeutic agents described below, one group will receive a bone marrow transplant, and one group will receive both chemotherapy and a bone marrow transplant. Subjects in each group will receive one or more of the following: VEGF-B[0283] ₁₆₇, VEGF-B₁₈₆, a VEGF-B N-acetylated variant, PDGF-A, PDGF-B, PDGF-C, and PDGF-D or a control protein. Before administering the compounds, blood samples are collected, for white blood cell characterization as described in Example 1, and additionally with anti-PDGF-receptor-α and anti-PDGF-receptor-β antibodies.
Chemotherapeutic agents for use include the following: cyclophosphamide (5,725 mg/m2), cisplatin (165 mg/m2), and carmustine (BCNU) (600 mg/m2)—to be administered over a four-day period. Three hours after marrow reinfusion, the administration of the particular growth factor product or control protocol is initiated as a continuous intravenous (IV) infusion for 14 to 21 days, or as a second dose schedule administered as a daily four-hour infusion for up to 21 days. Growth factor product or control protein is administered at dosages of 0.1 to 100 μg/kg/day. [0284]
In addition to blood sampling every 2-3 days (with characterization of white blood cells as described in Example 1, and additionally with anti-PDGF-receptor-α and anti-PDGF-receptor-β antibodies used), bone marrow biopsies and marrow progenitor assays are performed at five-day intervals, and the functional characteristics of white blood cells are monitored before, during, and, if abnormal, after infusion. [0285]

EXAMPLE 8

PDGF-CC Mobilizes Endothelial Progenitors Upon Tissue Ischemia In Vivo

To examine the possible mechanism by which PDGF-CC (PDGF-CC refers to a homodimer of PDGF-C) stimulates vessel growth and maturation, the effects of PDGF-CC on vascular endothelial progenitor cells (EPCs) were assayed. [0286]
For the EPC mobilization assay, mice were treated with PDGF-CC protein (4.5 μg/day: an approximation based on 30 μg per week) using subcutaneously implanted osmotic minipumps (Alzet, type 2001) immediately after femoral (hind limb) artery ligation. After two or five days, mice were sacrificed and spleens harvested for EPC analysis using procedures described previously [Dimmeler, S., et al., [0287] J. Clin. Invest. 108:391-97 (2001); Asahara, T., et al., Circ. Res. 85:221-8 (1999)]. Spleens were mechanically minced using syringe plungers and laid over Ficoll to isolate splenocytes. Splenocytes were seeded into fibronectin-coated 24-well plates in 0.5 ml EBM medium. After three weeks of culturing, adherent cells were stained for Dil-Ac-LDL/lectin and number of the positive cells counted. Late outgrowth EPCs (after 3 weeks of culture) were identified by metabolic uptake of DiI-acetylated-LDL (Molecular Probes) and positive staining of Alexa 488-labeled isolectin B4 (Molecular Probes). Quantification of the EPC density was performed by confocal microscopy in five microscopic fields at 200× magnification, and average EPC density calculated.
Specifically, EPC mobilization was quantified by counting the number of acLDL-DiI/isolectin-IB4 positive endothelial cells after 3 weeks of plating out spleen mononuclear cells. By scoring only after 3 weeks, only late-outgrowth EPCs, and not surviving sludged-off endothelial cells, are selectively assayed [Lin, Y., et al., [0288] J. Clin. Invest. 105:71-7. (2000); Rafii, S., J. Clin. Invest. 105:17-9 (2000)]. In baseline conditions, PDGF-CC did not affect mobilization of EPCs (EPCs/mm2: 139±26 in control versus 134+14 after PDGF-CC, n=6 each group, P=0.9, FIG. 4).
To look at the effect of PDGF-CC under ischemic conditions, femoral arteries were ligated. Treatment with PDGF-CC protein for two days (4.5 μg/day via minipump) in the mice augmented EPC mobilization approximately Four-fold above the levels found in the control group from [0289] day 2 to day 5 after hind limb ischemia (EPCs/mm2: 155±47 in control versus 641±207 after PDGF-CC, n=9,10, “*” P<0.05, FIG. 4; Values are presented as mean +\− SEM. of 10 mice.). This augmentation persisted to day five, albeit at a lower level (EPCs/mm²:249±42 in control versus 528±157 after PDGF-CC; n=10 each group, P=0.1, FIG. 4). Thus, PDGF-CC treatment enhanced EPC mobilization in tissue ischemia, thereby providing a source of ECs needed for revascularization of ischemic tissues.
PDGF-CC mediated EPC mobilization is an early and sustained event after tissue ischemia, starting at [0290] day 2 after ischemia and continuing onwards. This time window parallels the onset of ischemia-induced angiogenesis and thus leads to the possibility of an efficient launching of angiogenesis by providing sufficient amount of EPCs. The foregoing data demonstrate that PDGF-CC can be employed to mobilize EPCs at a time of active revascularization of ischemic tissues. In these experiments, PDGF-CC induced EPC mobilization was ischemia-dependent. EPC mobilization was increased by PDGF-CC in mice with hind limb ischemia, but not in normal ones, suggesting that PDGF-CC exerts its function in concert with other ischemia-dependent factors.
The hindlimb ligation and EPC migration assays described above were repeated using PDGF-AA, PDGF-BB, and PDGF-CC, as well as control vehicle. The model used was the Balb/c hind limb ischemia model as described in of Luttun, et al., [0291] Nat. Med. 8:831-40 (2002). The following modifications over the procedures described above were carried out: The concentration of the protein was 4.3 μg per day for each factor and the analysis was done after 2 days. The vehicle was PBS. An Alzet minipump 1003D, which works for two consecutive days was used instead of a minipump 2001, which works for seven consecutive days. The pump rate/drug delivery is exactly the same, and drug loading was calculated and performed in a way that it matched the 30 μg over 7 days strategy. EPC mobilization was assayed in ligated mice, at day 2 post-ligation; treatments continuously given via osmotic minipumps, blind analysis. EPC mobilization is expressed as density per mm²:vehicle (n=1): 119; PDGF-AA (n=7): 1052+/−177; PDGF-BB (n=8): 858+/−195; PDGF-CC (n=3): 1328+/−228. These results confirm those described above, and also demonstrate the superiority of PDGF-CC over PDGF-AA and PDGF-BB in mobilizing EPCs.

EXAMPLE 9

PDGF-CC Enhances Differentiation of Bone Marrow Progenitor Cells into Both Endothelial and Smooth Muscle Cells

Upon stimulation by growth factors or cytokines, bone marrow stem/progenitor cells can differentiate into ECs and SMCs and thereby contribute to angio/arteriogenesis [Orlic, D., et al., [0292] Nature 410:701-5 (2001); Kawamoto, A., et al., Circulation 103:634-7 (2001); Asahara, T., et al., Circ. Res. 85:221-8 (1999). The potential role of PDGF-CC in the differentiation of bone marrow stem/progenitor cells into vascular cells was investigated as follows.
A. Adherence Assay [0293]
To investigate this potential role of PDGF-CC, an adherence assay was first performed. Enriched human BM derived AC133+CD34+cells—a population enriched for stem/progenitor cells [Miraglia, S., et al., [0294] Blood 90:5013-21 (1997); Yu, Y., et al., AC133-2, J. Biol. Chem. 277:20711-6 (2002); Donnelly, D. S. & Krause, D. S., Leuk Lymphoma 40:221-34 (2001).]—(Clonetics) at 10⁵/ml were cultured for 3 days in HPGM (Clonetics) in a 6-well plate (Becton Dickinson). [Miraglia, S., et al., Blood 90:5013-21 (1997); Yu, Y., et al., AC133-2, J. Biol. Chem. 277:20711-6 (2002); Donnelly, D. S. & Krause, D. S., Leuk. Lymphoma 40:221-34 (2001)] Cells were then seeded in collagen coated 12-well plates in EBM (Clonetics) medium containing 4% FCS and VEGF₁₆₅(R&D Systems) or PDGF-CC (50 ng/ml each). These cells expressed PDGFR-A, when analyzed by RT-PCR (not shown). Growth factors were added every two days and media were refreshed at 75% every four days.
For the adherence assay, 2.5×10[0295] ⁴of non-adherent cells/ml were cultured in the same conditions on chamber slides coated with collagen, or in a 96-well plate coated with 0.3% gelatin in PBS. Cells were then washed three times with PBS, fixed and stained with May-Grünwald Giemsa (Sigma) after two weeks of culture on chamber slides (Becton Dickinson). The number of viable cells were estimated by ATP quantification using cellTiter-glo luminescent cell viability assay (Promega) according to the manufacturer's instructions.
After two weeks of stimulation, both PDGF-CC and VEGF enhanced the cell adherence, a prerequisite for anchorage-dependent cell proliferation, differentiation, migration and prevention of apoptosis [Assoian, R. K. [0296] J Cell Biol 136, 1-4. (1997); Asahara, T. et al. Science 275, 964-7. (1997)] (FIG. 5; *: P<0.05. Values are presented as mean+/−SEM.)
B. Cell Differentiation Assays [0297]
The cell differentiation assays involved cell surface marker staining, cells (2×10[0298] ⁴/well) cultured on collagen-coated culture slides for two, three, and four weeks were fixated (45 min, 25° C.) and permeabilized (45 min, 25° C.) using a Intrastain Kit (DAKO), and then labeled with CD31 FITC (Becton Dickinson), CD144 FITC (Pharmingen), CD34 FITC (Becton Dickinson) or SMC-Actin CY3 (Sigma). Single or double-labeled cells were analyzed using laser confocal immunofluorescence microscopy. The same kinds of cells and conditions as described in part “A” for the adherence assay were also used for the cell differentiation assays.
The results showed that PDGF-CC and VEGF markedly differed in their ability to induce the commitment of these stem cells into either the endothelial or smooth muscle cell lineages. After two weeks of stimulation, both PDGF-CC and VEGF induced the expression of EC surface markers CD 144 (VE-cadherin) and CD31 (PECAM)), indicating that these growth factors induced a characteristic endothelial phenotype. Vehicle-treated (control) cells remained negative for these markers. Only PDGF-CC additionally induced the expression of the smooth muscle cell marker SMA in a fraction of these cells, indicating that these cells had acquired a characteristic SMC phenotype. The VEGF-treated cells did not become SMA positive relative to background (nor did controls). Double labeling experiments revealed that PDGF-CC often induced the expression of CD31 and SMA in the same cells. [0299]
By four weeks, most (>95%) of the PDGF-CC-treated cells were SMA positive and had lost their expression of CD144 and CD31. In contrast, VEGF-treated cells were still CD144 and CD31 positive and remained SMA negative. Thus, PDGF-CC initially induced bone marrow progenitor cells to differentiate into cell types with both endothelial or smooth muscle cell characteristics—eventually, after long-term treatment, yielding cells with a SMC-like phenotype. PDGF-CC thus differed from VEGF, as the latter only caused bone marrow progenitors to acquire EC-specific markers, even after prolonged treatment. [0300]
Continuing the discussion of the significance of the finding that PDGF-CC mobilizes vascular stem/progenitor cells, increases stem cell adherence/viability, and promotes stem cell differentiation is the present discovery that PDGF-CC mediated BM cell differentiation is bi-directional, that is, both EPC- and SMC-oriented. The final destination of the stem cells probably depends on the cellular environment, and needs to be co-orchestrated by other growth factors or cytokines. In the presence of VEGF, which often is a sign of tissue ischemia, the BM cells may be better directed to their EC fate and contribute to the initial stage of angiogenesis-capillary formation. PDGF-CC may further strengthen the second stage of angiogenesis-vessel maturation, by providing SMCs to the capillaries and leading to a stabilized functional vasculature. Without high levels of VEGF, that is, in normoxia, PDGF-CC turns ultimately the BM cells into SMCs, thus avoiding the possibility of angioma-genesis (Angioma-genesis is discussed in Carmeliet, [0301] P. Nat Med 6, 1102-3. (2000).). Taken together, the early and ischemia-dependant EPC mobilization and the bi-directional BM cell differentiation conferred by PDGF-CC provide a valuable characteristic of both efficiency and safety for the growth factor's in vivo therapeutic usage in building new blood vessels to treat ischemic diseases.
Moreover, the angio/arteriogenic effect of PDGF-CC involves several mechanisms, including mobilization and differentiation of vascular progenitors, chemotactic effect on differentiated both ECs and SMCs, proliferation and migration of perivascular cells, and upregulation of VEGF expression. Thus, in contrast to VEGF or PDGF-AA and -BB, whose vascular effects are largely restricted to EC or SMC/fibroblast cells, respectively, the effect of PDGF-CC on the vasculature is more pleiotropic and thus allows for a more synchronized, universal action of the different cell types, needed to build functional blood vessels. [0302]
The abilities of PDGF-CC to mobilize vascular progenitors, by promoting their differentiation into both endothelial and smooth muscle cells, and stimulate these differentiated vascular cells, indicate that PDGF-CC is useful in vitro and in vivo orchestrating the complex process of building mature, durable and functional vessels. [0303]

EXAMPLE 10

PDGF-CC Promotes Endothelial Cell Migration and Microvessel Sprouting

In this example, the effect of PDGF-CC on EC migration and proliferation was compared to that of VEGF (which primarily affects endothelial cells [Senger, D. R., et al., [0304] Am. J. Pathol. 149:293-305. (1996)]) and PDGF-AA and -BB (which primarily affect fibroblasts and smooth muscle cells [Heldin, C. H. & Westermark, B. Physiol. Rev. 79:1283-1316 (1999)]). Migration, proliferation and aortic ring assays were performed.
A. Cell Migration Assays [0305]
Cell migration assays were performed on growth-arrested confluent HMVEC or BAEC cells. Cell monolayers were wounded with a rubber policeman and washed with serum-free medium. Dishes were then incubated for 20 hours in serum-free medium containing VEGF165, PDGF-AA, -BB (R&D Systems, Minneapolis USA) or PDGF-CC. Each assay included two dishes per condition and was repeated three times independently. Cells were photographed at 40× magnification, and migration percentage corresponding to the ratio between area of the cells and the total area of the wound (Biocom visiol@b 2000 version 4.52, San Diego). For the cell migration assay, ANOVA Dunett's test was used for data analyzing, with P<0.05 considered statistically significant. Data are presented as mean+\−SEM. [0306]
PDGFR-α expression on the human microvascular endothelial cells (HMVEC) was confirmed by Western blot, albeit at a lower level as compared with that of the SMCs (not shown). VEGF and PDGF-CC, but not PDGF-AA or PDGF-BB, stimulated migration of human microvascular endothelial cells (HMVEC) and bovine aorta endothelial cells (BAEC) (FIG. 6[0307] a).
B. Proliferation Assay [0308]
For HMVEC proliferation assay, cells were seeded in 96-well plates (5 wells per condition), and incubated with PDGF-AA, PDGF-BB or PDGF-CC (50 ng/ml) after serum starvation. After 7 days, viable cells were counted using cellTiter-glo luminescent cell viability assay (Promega). For NIH-3T3 and hSMC proliferation assay, cells cultured in 96-well plates were serum-starved overnight, followed by treatment with growth factors at different concentrations. Two days later, cell numbers were counted and proliferation percentage calculated, using cells cultured in medium containing 10% serum as control. [0309]
In contrast to the migration results, none of the PDGFs affected EC proliferation (FIG. 6[0310] b), in agreement with the previous observation that PDGFR-A does not transmit mitogen signals in ECs [Marx, et al., J. Clin. Invest. 93:131-9 (1994)], whereas VEGF dramatically induced EC proliferation (FIG. 6b; *: P<0.05. Values are presented as mean+/−SEM.).
C. Aortic Ring Assay [0311]
The aortic ring assay is a means of assessing outgrowth of microvessels from an intact vessel in vitro [Blacher, S., et al., [0312] Angiogenesis 4:133-42 (2001)]. The assay was performed as described in [Blacher, S., et al., Angiogenesis 4:133-42 (2001)]. Briefly, one-millimeter long aortic rings were embedded in gels of rat tail interstitial collagen and cultured at 37° C., supplemented with different growth factors (50 ng/ml). Experiments included three explants per condition and were repeated at least twice. Aortic rings were photographed at 25× magnification.
At [0313] day 9 after culturing, microvessels and the distance of their outgrowth from the aortic ring were quantified and evaluated using Student's t-test. Specifically, two-tailed Student's t-test was used for data analysis, with P<0.05 considered statistically significant. For cell migration assay, ANOVA Dunett's test was used for data analyzing, with P<0.05 considered statistically significant. Quantification of the outgrowth of microvascular sprouts and perivascular fibroblast-like cells was performed using computer-assisted morphometry.
In baseline conditions, only a small number of microvessels sprouted from the aortic rings—most of them over very short distances (0.25 mm from the aortic ring) and only a small fraction (<5%) growing out over longer distances (>0.5 mm from the aortic ring). VEGF had the most potent effect on microvessel outgrowth. VEGF not only increased the number of sprouting microvessels (P<0.001 at all concentrations versus control), but also the distance over which they grew out (P<0.05 at all concentrations versus control; FIG. 7[0314] a, b).
PDGF-CC enhanced the outgrowth of both microvascular sprouts and fibroblast-like cells. At 5-10 ng/ml, PDGF-CC maximally stimulated perivascular fibroblast-like cells, which emigrated over much greater distances from the aortic ring. At high concentrations (30-50 ng/ml), PDGF-CC still stimulated fibroblast-like cell growth and emigration, but less significantly than at lower concentrations, possibly because the perivascular cells were recruited by the sprouting microvessels. PDGF-CC at 30 ng/ml increased the number of microvessels (P<0.001 versus control, FIGS. 7[0315] a, 7 b) and increased the distance of vessel outgrowth at 5 ng/ml (P<0.01 versus control, FIGS. 7a, 7 b). Unlike VEGF, which was ineffective on perivascular fibroblast-like cells, PDGF-CC increased the number and migration of the perivascular cells over much greater distances from the aortic ring, while PDGF-AA has an intermediate effect. Apparently, PDGF-CC had its maximum effect at 30 ng/ml on microvessel sprouting, and was less potent at a concentration of 50 ng/ml, indicating that the dose-response relationship of PDGF-CC in the aortic ring assay was bell-shaped. A similar bell-shaped dose-response relationship has been documented for other members of the VEGF/PDGF-superfamily [Jin, K. L., et al., J. Mol. Neurosci. 14:197-203 (2000)].
PDGF-AA and -BB had no effect on the number of microvessels (FIG. 7[0316] a), although they both increased the distance of vessel outgrowth at different concentrations (5 ng/ml for PDGF-AA and 20-50 ng/ml for PDGF-BB respectively, P<0.01 versus control, FIG. 7a). Thus, PDGF-CC mobilized EC migration in cultured cells and promoted microvessel sprouting in aortic ring assay. This chemotactic effect of PDGF-CC on ECs is surprising, because although the other PDGFs are among the most potent stimuli of mesenchymal cell migration, they either do not or only minimally stimulate EC migration. In certain conditions, they even inhibit EC migration. [Thommen, J Cell Biochem. 1997 Mar. 1;64(3):403-13; De Marchis, F., et al., Blood 99:2045-2053 (2002)]

EXAMPLE 11

PDGF-CC is Both Chemotactic and Mitogenic for Smooth Muscle Cells and Perivascular Fibroblast Cells

This example describes the mitogenic and chemotactic effects of PDGF-CC on SMCs and perivascular fibroblast cells, and compared the effect of PDGF-CC on such cells in different cellular environments—in both cultured cells and aortic ring assay, in comparison with VEGF, PDGF-AA and PDGF-BB. [0317]
A. Cell Migration Assay [0318]
Cell migration assays were performed as described in Example 10. In cell culture assay, all three PDGFs stimulated hSMCs migration with a comparable potency, while VEGF had no effect on SMC migration (FIG. 6[0319] a). Thus, interestingly, PDGF-CC promoted the migration of both ECs and SMCs, while VEGF only stimulated EC migration, and PDGF-AA, -BB only SMCs. This observation is consistent with the aortic ring assay, where PDGF-CC stimulated microvessel outgrowth while PDGF-AA and -BB were less effective.
B. Aortic Ring Assay [0320]
In the aortic ring assay (assay described in Example 10), the growth and emigration of perivascular fibroblasts from the intact vessel was quantified using computer-assisted image analysis after treatment with different PDGFs at different concentrations. [0321]
In baseline conditions, individual perivascular fibroblasts (identified as isolated cells, not associated with sprouting microvessels) were sparse and emigrated over only short distances from the aortic ring. PDGF-CC promoted the proliferation and migration of the fibroblast-like perivascular cells dramatically at all different concentrations tested, with an optimum concentration of 5-10 ng/ml. The mitogenic effect of PDGF-CC was much greater than those of PDGF-AA and -BB. VEGF had no mitogenic activity on the fibroblast-like cells. PDGF-CC significantly increased the number of fibroblasts, which also emigrated over much greater distances from the aortic ring (P<0.001 at all concentrations versus control, FIG. 7[0322] b). At high concentrations (30-50 ng/ml), PDGF-CC still stimulated fibroblast growth and emigration but less significantly than at lower concentrations, possibly because its effects were dose-dependent (see above) and/or the perivascular cells surrounded the sprouting microvessels. PDGF-AA had an intermediate effect (P<0.05 at different concentrations versus control, FIG. 7b). In contrast, VEGF had no and PDGF-BB only had a effect at a concentration of 50 ng/ml on perivascular fibroblast growth and emigration (P<0.05 in PDGF-BB versus control, FIG. 7b). Thus, of all PDGF homologues, PDGF-CC most significantly stimulated migration and proliferation of perivascular cells in the aortic ring assay—an assay that is believed to reflect more closely the in vivo situation and allows synergistic interactions between the different vascular cell types [Hartlapp, I. et al, Faseb J, 2001, 15: 2215-24; Blacher, S., et al. Angiogenesis 4:133-42 (2001); Nehls, V., et al., Cell Tissue Res. 270:469-74 (1992); Tille, J. C. & Pepper, M. S., Exp. Cell. Res. 280:179-91. (2002)]
C. Western Blot and Receptor Activation Assays [0323]
For Western blot assay, subconfluent cells were rinsed with cold PBS supplemented with 5 g/ml of antiprotease cocktail, lysed in RIPA buffer and analyzed on 10% acrylamide SDS PAGE in reducing condition. Two antibodies to PDGFR-α (rabbit polyclonal antibody, dilution: 1/500, Santa Cruz, sc431; and monoclonal peroxidase-labeled anti-rabbit antibody, dilution: 1/2500, Sigma, A-2074) were used for protein detection. Membranes were developed using the Supersignal System (Pierce). For receptor activation, and tissue/cell lysates were subjected to immunoprecipitation using the rabbit anti-PDGFR-α antibody. The precipitants were analyzed on SDS-PAGE, and immunoblotted using a monoclonal anti-phosphotyrosine antibody (PY99, Santa Cruz). PDGF-CC induced proliferation of hSMC and NIH3T3 cells, but not ECs. In cultured hSMC and NIH-3T3 fibroblast cells, in which PDGFR-α is highly expressed and activated (FIG. 8A; PDGFR-α was highly expressed (Western blot, upper lanes—anti-PDGFR-α) and activated/phosphorylated (lower lanes—anti-phosphotyrosine) in the hSMC and NIH-3T3 cells.). In cell culture system, PDGF-CC induced the proliferation of hSMC and NIH-3T3 fibroblast cells. All three PDGFs displayed about the same degree of mitogenic activity—with the effect of PDGF-CC on hSMC cells being slightly more pronounced. (FIG. 8[0324] b.)

EXAMPLE 12

PDGF-CC Upregulates VEGF Expression

Because the foregoing data indicates that PDGF-CC induced some VEGF-like effects, the ability of PDGF-CC to upregulate the expression of VEGF was examined. The initial results (of infarcted tissue using LAD ligation) suggested such an effect as they showed more prominent VEGF immunoreactivity in the border zones surrounding the infarcts after PDGF-CC treatment than control. [0325]
To further confirm the initial results, PDGF-C was overexpressed in NIH-3T3 fibroblast cells and VEGF expression was measured at both RNA and protein levels. For PDGF-C over-expression, mouse full-length PDGF-C cDNA was cloned into pcDNA3.1/zeo(+) mammalian expression vector (Invitrogen) and the construct was verified by sequencing. Plasmid DNA was transfected into semiconfluent cells using Lipofectamine plus reagent according to manufacturers protocol (Life technology). Stable transfectants were selected with 700 μg ml-1 Zeocin (Invitrogen) for 3 weeks. Resistant colonies were pooled and maintained in medium supplemented with 300 μg ml-1 Zeocin. [0326]
Over-expression of PDGF-C was confirmed by Western blotting (FIG. 8[0327] c, lower-left panel). For PDGF-CC Western blot assay, cells were starved in serum-free medium overnight. Conditioned media (overnight) were collected and protein concentration determined (Bradford, 1976). 35 μg of protein was trichloroacetic acid (TCA) precipitated and subjected to Western blot using affinity purified polyclonal rabbit antibodies against PDGF-CC [Li, et al., Nat. Cell. Biol. 2:302-09 (2000)]. All the samples were in triplicates and the experiment was repeated twice. Secreted VEGF protein was quantified using the Quantikine immunoassay kit (R&D system) according to the manufacturers protocol.
RNase protection analysis (RPA) was performed according to the manufacturer's protocol (Ambion) to investigate gene expressions at mRNA level. Riboprobes were prepared using RNA polymerase (Promega) and 32P-UTP (Amersham). Mouse β-actin cDNA (250 bp, Ambion) was used as an internal control. VEGF mRNA level was significantly upregulated in the PDGF-CC over-expressing (NIH-3T3) cells as compared to that of vector transduced cells by RPA assay (FIG. 8C, upper-left panels). [0328]
ELISA assay further confirmed that secreted VEGF protein level in the serum-free PDGF-CC over-expressing cell-conditioned media was significantly increased as compared with that of the vector-transduced cell conditioned (mock-transfected cell conditioned) media (VEGF in pg/ml: 1140±96 in PDGF-CC versus 585±80 in control, n=6, P<0.01, FIG. 8C, right panel. *: P<0.05. Values are presented as mean+/−SEM.). The activity of PDGF-CC to upregulate VEGF may explain, at least in part, some of its angiogenic activities. [0329]

EXAMPLE 13

PDGF-CC Stimulates Angiogenesis and Arteriogenesis in the Ischemic Heart

A previously established mouse model of myocardial ischemia was used to assess whether PDGF-CC is capable of stimulating the revascularization of ischemic myocardium. After coronary ligation, new vessels revascularize the ischemic core from its surrounding border region. [0330]
RNAse protection analysis revealed that PDGFR-α transcripts for the PDGF-C receptor (PDGFR-α) were detectable in the normal myocardium. β-actin was used as an internal control. Moreover, immunoprecipitation and subsequent Western blotting using an equal amount of protein extract revealed that PDGFR-α protein levels were significantly upregulated in the ischemic border zones surrounding the infarcts, i.e., where vessel growth is most active, as compared to the rest of the normal myocardium. PDGFR-α was activated more in the border zones than in the normal (non-ischemic) regions of the heart, and maximally after PDGF-CC treatment. PDGFR-α was, as assessed by Western blotting of the phosphorylated tyrosine residues after immunoprecipitation, highly activated in the border zone surrounding the infarcts. [0331]
Acute myocardial ischemia and hind limb ischemia mouse models that have been were previously described [Luttun, A. et all, [0332] Nat. Med. 8:831-40 (2002).; Heymans, S. et al. Nat Med 5, 1135-42 (1999).] were used in experiments. Subcutaneously implanted osmotic minipumps (Alzet, type 2001) were used for continuous protein delivery for 7 days. Human PDGF-CC core domain protein was produced as described. [Li, X. et al. Nat Cell Biol 2, 302-309 (2000).] Fluorescent or color dye microspheres (yellow, 15 μm, Molecular Probes) were administered after maximal vasodilatation (sodium nitroprusside, 50 ng/ml, Sigma) for blood flow measurement, and flow was calculated as described. [Carmeliet, P. et al. Nat Med 5, 495-502. (1999).] For histology, the hearts were harvested seven days after LAD (left anterior descending coronary artery) ligation, and sectioned longitudinally (6 μm). Infarcted areas were morphologically inspected after immunohistochemistry staining using thrombomodulin (rabbit anti-TM, for all vessels) and smooth muscle alpha-actin (mouse anti-SMA, for mature SMC covered vessels, Dako), and vessel densities calculated. Gastrocnemius muscles after femoral artery ligation were sectioned transversally and analyzed after H&E or immunostainings with the EC marker CD31 (PECAM, rat anti-CD31, Pharmingen). Vessel densities and tissue necrosis/regeneration in the gastrocnemius muscle were analyzed morphmometrically using the KS300 image analysis soft ware (Zeiss). Remodeling of collateral vessels in the upper hind limb after femoral ligation was quantified as reported. [Luttun, A. et al. Nat. Med. 8:831-40 (2002).] Rabbit polyclonal VEGF antibody (Santa Cruz, sc-152) was used for immunohistochemistry staining.
To examine whether PDGF-CC could stimulate revascularization of the ischemic myocardium recombinant human PDGF-CC core domain protein was delivered using a minipump, continuously over one week after coronary ligation. PDGF-CC protein treatment increased vascular density in the infarcted areas in a dosage dependent way. Compared to control, PDGF-CC also increased the amount of active PDGFR-α in the border region. After seven days, angiogenesis was quantified by counting the number of endothelial cell (EC)-lined vessels in the ischemic area after immunolabeling with thrombomodulin (TM). Vessel maturation (arteriogenesis) was evaluated by counting the arterioles, immunoreactive for smooth muscle cell β-actin (SMA). At 1.5 μg/day, PDGF-CC minimally affected the TM-positive vessel density (vessels/mm2: 175±8 in control, n=21 versus 190+13 after 10 μg PDGF-CC, n=7; P═NS), but increased, by 1.36-fold, the number of SMA-positive arterioles (vessels/mm2: 40±9 in control, n=21 versus 54±4 after 10 μg PDGF-CC; n=7; P<0.05). When using a 3-fold higher dose (4.5 μg/day), PDGF-CC significantly stimulated angiogenesis (TM-positive vessels/mm2: 175±8 in control versus 230±22 after PDGF-CC; n=10-21; P<0.05) and arteriogenesis (SMA-positive vessels/mm2: 40±9 in control versus 58+7 after PDGF-CC; n=10-21; P<0.05). [0333]
No signs of hemorrhage, edema or fibrosis were observed in the PDGF-CC treated hearts. These new vessels were functional as perfusion of the ischemic myocardial region was significantly increased by 1.4-1.7 fold (blood flow in ml/min/g: infarct: 1.6±0.2 in control versus 2.2±0.2 after 30 μg PDGF-CC; n=7-9; P<0.05; normal part of the infarcted heart: 2.0±0.2 in control versus 3.3±0.5 after 30 μg PDGF-CC; n=7-9; P<0.05. Blood flow such that 30 μg is approx. to be 4.5 μg/day: 1.6+0.2 ml/min/g in control versus 2.2±0.2 ml/min/g after PDGF-CC; n=7-9; P<0.05). The effect of PDGF-CC to stimulate revascularization appeared to be restricted to the ischemic heart, as no differences were observed in vessel density in other organs (average blood flow of left and right kidney in ml/min/g: 5.2±0.4 in control versus 5.7±0.5 after 30 μg PDGF-CC; n=7-9; P=0.5). [0334]
The magnitude of revascularization of the ischemic myocardium induced by PDGF-CC is comparable to that of VEGF and PlGF. The mice tolerated the PDGF-CC treatment without problems, appeared healthy and had no signs of toxicity (weight loss, inactivity). Thus, PDGF-CC protein treatment promoted functional revascularization in cardiac ischemia via enhanced angiogenesis (more vessels) and arteriogenesis (more SMC coverage). The angio/arteriogenic activity of PDGF-CC in cardiac ischemia is surprising, because the other PDGFR-α ligand, PDGF-AA is poorly angiogenic or even suppresses angiogenesis. [0335]

EXAMPLE 14

Therapeutic Angiogenesis with PDGF-CC in Ischemic Limbs PDGF-CC Stimulates Angiogenesis in the Ischemic Limb

To further verify the angio/arteriogenic activity of PDGF-CC in vivo, the effect of PDGF-CC in an established mouse model of hind limb ischemia was also investigated. For the model see Luttun, A. et al. [0336] Nat. Med. 8:831-40 (2002). For more on limb ischemia, a common disease in humans see J Control Release 78, 285-94. (2002); Beckman, J. A., Creager, M. A. & Libby, P. Jama 287, 2570-81. (2002). PDGFR-α expression was first quantified by RNAse protection analysis, using β-actin as an internal control (ratio of PDGFR-A levels were normalized to the β-actin control), in the gastrocnemius muscle, which becomes highly ischemic after ligation of the femoral artery. [Deindl, E. et al. Circ Res 89, 779-86. (2001); Couffinhal T et al. American Journal of Pathology 152, 1667-1679 (1998).] Two days after femoral artery ligation, when a fraction of myocytes died due to ischemic necrosis, PDGFR-α transcript levels decreased to 76% of those found in normal muscles (PDGFR-α/β-actin transcript levels: 1.27+/−0.06 in normal muscle versus 0.96+/−0.05 after ligation, n=9, 10; P<0.01). However, compared to vehicle, a daily treatment with 4.5 μg PDGF-CC upregulated PDGFR-A expression at day 2 after ligation and almost completely restored its expression levels to those found in the unligated control muscle (PDGFR-α/β-actin transcript levels: 1.16+0.08 after PDGF-CC versus 0.96±0.05 in untreated, n=10 each group; P<0.05).
Revascularization of the ischemic gastrocnemius muscle, which only occurred in those regions where regenerating muscle replaced the necrotic avascular muscle, was scored after continuous delivery, by osmotic minipump, of 4.5 μg PDGF-CC per day for one week after femoral artery ligation. PDGF-CC protein treatment increased the PECAM+ capillary and SMA+ arteriolar density in the ischemic gastrocnemius muscles. PDGF-CC protein treatment decreased muscle necrosis and increased muscle regeneration in the gastrocnemius muscle at seven days after femoral artery ligation. Necrotic muscle fibers were identified as ghost cells lacking nuclei and containing a hyaline cytosol; regenerating myocytes were identified as small cells with central nuclei. Areas are expressed as percentage of the total muscle area. Treatment with PDGF-CC after femoral artery ligation not only increased angiogenesis (e.g. the capillary density; PDGFR-α/β-actin transcript levels: 1.16±0.08 after PDGF-CC versus 0.96±0.05 in untreated, n=10 each group; P<0.05), it also enhanced arteriogenesis (e.g. the density of SMA+vessels; SMA positive vessels/mm2: 53.1+/−3.7 after PDGF-C vs 38.6+/−4.8 after saline, n=15,16, P=0.02.). Moreover, PDGF-CC enhanced skeletal muscle regeneration (regenerating/total muscle area: 14±3% in control versus 27±4% after PDGF-CC, n=15, 16, P<0.05) and, as a result, also reduced the extent of ischemic muscle necrosis (necrotic/total muscle area: 80±3% in control versus 65+5% after PDGF-CC, n=15, 16, P<0.05;), suggesting that muscle regeneration and angiogenesis might be linked. PDGF-CC also enlarged the second-generation collateral side branches in the adductor muscle (680±40 μm[0337] ³after saline versus 920±100 μm³after PDGF-CC; N=10; P=0.05). No signs of hemorrhage, edema or fibrosis were observed in the PDGF-CC treated limbs. Muscle regeneration was maximal at sites of intense angiogenesis, suggesting that both processes were linked. PDGF-CC minimally affected the remodeling of the collateral vessels in the adductor muscle, presumably because of potential ischemia-dependant effect of PDGF-CC and this region is not ischemic after femoral artery ligation [Deindl, et al. Circ Res 89, 779-86 (2001); Pu, et al., J Invest Surg., 7(1): 49-60 (1994)]. Thus, PDGF-CC stimulates revascularization in mouse models of both heart and limb ischemia.
PDGF-CC was found to increase the perfusion of the ischemic myocardium by revascularizing the myocardium not only with SMC-covered coronary vessels (providing bulk flow) but also with endothelial-lined capillaries (distributing the flow to the individual cardiomyocytes). In the ischemic limb, PDGF-CC was also found to stimulate both angiogenesis and arteriogenesis. Moreover, the observation that PDGF-CC also enhanced muscle regeneration in areas of active revascularization further underscores that the new vessels were functional and perfused. The pleiotropic activity of PDGF-CC may also explain why no side effects of hemangioma-genesis and edema formation after PDGF-CC treatment were observed, which has been observed after VEGF administration. [0338]
PDGF-CC treatment mobilized endothelial progenitors and increased the vessel density and blood perfusion in the ischemic heart and limb, but did not affect quiescent vessels in other organs. Although PDGF-CC enlarged the second-generation side branches of the collateral vessels in the adductor muscle, this growth factor has, overall, a less dramatic effect on the remodeling of the preexisting collaterals in the upper limb region after femoral artery ligation than, for instance, bFGF, PlGF or GM-CSF. However, the molecular and cellular mechanisms of the growth of collateral vessels are quite distinct from those determining the formation of new capillaries and their maturation by coverage with smooth muscle cells. In particular, not ischemia but shear stress-induced recruitment of monocytes/macrophages is well known to play a critical role in initiating collateral growth in the upper hindlimb and PDGF-CC does not affect their recruitment (data not shown). Because only the lower, but not the upper limb is ischemic after femoral artery ligation, PDGF-C seems to be involved more in ischemia-dependent angiogenesis than in the shear stress-induced collateral remodeling. [0339]
Muscle regeneration was improved after femoral artery ligation by PDGF-CC, especially in regions where vascular regeneration was also maximal. [0340]
The foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting. Because modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof. [0341]
1 30 1 570 DNA Homo sapiens 1 accatgagcc ctctgctccg ccgcctgctg ctcgccgcac tcctgcagct ggcccccgcc 60 caggcccctg tctcccagcc tgatgcccct ggccaccaga ggaaagtggt gtcatggata 120 gatgtgtata ctcgcgctac ctgccagccc cgggaggtgg tggtgccctt gactgtggag 180 ctcatgggca ccgtggccaa acagctggtg cccagctgcg tgactgtgca gcgctgtggt 240 ggctgctgcc ctgacgatgg cctggagtgt gtgcccactg ggcagcacca agtccggatg 300 cagatcctca tgatccggta cccgagcagt cagctggggg agatgtccct ggaagaacac 360 agccagtgtg aatgcagacc taaaaaaaag gacagtgctg tgaagccaga cagccccagg 420 cccctctgcc cacgctgcac ccagcaccac cagcgccctg acccccggac ctgccgctgc 480 cgctgccgac gccgcagctt cctccgttgc caagggcggg gcttagagct caacccagac 540 acctgcaggt gccggaagct gcgaaggtga 570 2 188 PRT Homo sapiens mat_peptide (22)..(188) 2 Met Ser Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln Leu -20 -15 -10 Ala Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro Gly His Gln -5 -1 1 5 10 Arg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln 15 20 25 Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met Gly Thr Val 30 35 40 Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly Gly 45 50 55 Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln 60 65 70 75 Val Arg Met Gln Ile Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly 80 85 90 Glu Met Ser Leu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys 95 100 105 Lys Asp Ser Ala Val Lys Pro Asp Ser Pro Arg Pro Leu Cys Pro Arg 110 115 120 Cys Thr Gln His His Gln Arg Pro Asp Pro Arg Thr Cys Arg Cys Arg 125 130 135 Cys Arg Arg Arg Ser Phe Leu Arg Cys Gln Gly Arg Gly Leu Glu Leu 140 145 150 155 Asn Pro Asp Thr Cys Arg Cys Arg Lys Leu Arg Arg 160 165 3 624 DNA Homo sapiens 3 atgagccctc tgctccgccg cctgctgctc gccgcactcc tgcagctggc ccccgcccag 60 gcccctgtct cccagcctga tgcccctggc caccagagga aagtggtgtc atggatagat 120 gtgtatactc gcgctacctg ccagccccgg gaggtggtgg tgcccttgac tgtggagctc 180 atgggcaccg tggccaaaca gctggtgccc agctgcgtga ctgtgcagcg ctgtggtggc 240 tgctgccctg acgatggcct ggagtgtgtg cccactgggc agcaccaagt ccggatgcag 300 atcctcatga tccggtaccc gagcagtcag ctgggggaga tgtccctgga agaacacagc 360 cagtgtgaat gcagacctaa aaaaaaggac agtgctgtga agccagacag ggctgccact 420 ccccaccacc gtccccagcc ccgttctgtt ccgggctggg actctgcccc cggagcaccc 480 tccccagctg acatcaccca tcccactcca gccccaggcc cctctgccca cgctgcaccc 540 agcaccacca gcgccctgac ccccggacct gccgccgccg ctgccgacgc cgcagcttcc 600 tccgttgcca agggcggggc ttag 624 4 207 PRT Homo sapiens mat_peptide (22)..(207) 4 Met Ser Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln Leu -20 -15 -10 Ala Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro Gly His Gln -5 -1 1 5 10 Arg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln 15 20 25 Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met Gly Thr Val 30 35 40 Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly Gly 45 50 55 Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln 60 65 70 75 Val Arg Met Gln Ile Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly 80 85 90 Glu Met Ser Leu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys 95 100 105 Lys Asp Ser Ala Val Lys Pro Asp Arg Ala Ala Thr Pro His His Arg 110 115 120 Pro Gln Pro Arg Ser Val Pro Gly Trp Asp Ser Ala Pro Gly Ala Pro 125 130 135 Ser Pro Ala Asp Ile Thr His Pro Thr Pro Ala Pro Gly Pro Ser Ala 140 145 150 155 His Ala Ala Pro Ser Thr Thr Ser Ala Leu Thr Pro Gly Pro Ala Ala 160 165 170 Ala Ala Ala Asp Ala Ala Ala Ser Ser Val Ala Lys Gly Gly Ala 175 180 185 5 13 PRT Artificial sequence Synthetic peptide 5 Pro Xaa Cys Val Xaa Xaa Xaa Arg Cys Xaa Gly Cys Cys 1 5 10 6 2108 DNA Homo sapiens misc_feature (2002) n = a, c, g, or t 6 ccccgccgtg agtgagctct caccccagtc agccaaatga gcctcttcgg gcttctcctg 60 gtgacatctg ccctggccgg ccagagacga gggactcagg cggaatccaa cctgagtagt 120 aaattccagt tttccagcaa caaggaacag aacggagtac aagatcctca gcatgagaga 180 attattactg tgtctactaa tggaagtatt cacagcccaa ggtttcctca tacttatcca 240 agaaatacgg tcttggtatg gagattagta gcagtagagg aaaatgtatg gatacaactt 300 acgtttgatg aaagatttgg gcttgaagac ccagaagatg acatatgcaa gtatgatttt 360 gtagaagttg aggaacccag tgatggaact atattagggc gctggtgtgg ttctggtact 420 gtaccaggaa aacagatttc taaaggaaat caaattagga taagatttgt atctgatgaa 480 tattttcctt ctgaaccagg gttctgcatc cactacaaca ttgtcatgcc acaattcaca 540 gaagctgtga gtccttcagt gctaccccct tcagctttgc cactggacct gcttaataat 600 gctataactg cctttagtac cttggaagac cttattcgat atcttgaacc agagagatgg 660 cagttggact tagaagatct atataggcca acttggcaac ttcttggcaa ggcttttgtt 720 tttggaagaa aatccagagt ggtggatctg aaccttctaa cagaggaggt aagattatac 780 agctgcacac ctcgtaactt ctcagtgtcc ataagggaag aactaaagag aaccgatacc 840 attttctggc caggttgtct cctggttaaa cgctgtggtg ggaactgtgc ctgttgtctc 900 cacaattgca atgaatgtca atgtgtccca agcaaagtta ctaaaaaata ccacgaggtc 960 cttcagttga gaccaaagac cggtgtcagg ggattgcaca aatcactcac cgacgtggcc 1020 ctggagcacc atgaggagtg tgactgtgtg tgcagaggga gcacaggagg atagccgcat 1080 caccaccagc agctcttgcc cagagctgtg cagtgcagtg gctgattcta ttagagaacg 1140 tatgcgttat ctccatcctt aatctcagtt gtttgcttca aggacctttc atcttcagga 1200 tttacagtgc attctgaaag aggagacatc aaacagaatt aggagttgtg caacagctct 1260 tttgagagga ggcctaaagg acaggagaaa aggtcttcaa tcgtggaaag aaaattaaat 1320 gttgtattaa atagatcacc agctagtttc agagttacca tgtacgtatt ccactagctg 1380 ggttctgtat ttcagttctt tcgatacggc ttagggtaat gtcagtacag gaaaaaaact 1440 gtgcaagtga gcacctgatt ccgttgcctt gcttaactct aaagctccat gtcctgggcc 1500 taaaatcgta taaaatctgg attttttttt ttttttttgc tcatattcac atatgtaaac 1560 cagaacattc tatgtactac aaacctggtt tttaaaaagg aactatgttg ctatgaatta 1620 aacttgtgtc rtgctgatag gacagactgg atttttcata tttcttatta aaatttctgc 1680 catttagaag aagagaacta cattcatggt ttggaagaga taaacctgaa aagaagagtg 1740 gccttatctt cactttatcg ataagtcagt ttatttgttt cattgtgtac atttttatat 1800 tctccttttg acattataac tgttggcttt tctaatcttg ttaaatatat ctatttttac 1860 caaaggtatt taatattctt ttttatgaca acttagatca actattttta gcttggtaaa 1920 tttttctaaa cacaattgtt atagccagag gaacaaagat ggatataaaa atattgttgc 1980 cctggacaaa aatacatgta tntccatccc ggaatggtgc tagagttgga ttaaacctgc 2040 attttaaaaa acctgaattg ggaanggaan ttggtaaggt tggccaaanc ttttttgaaa 2100 ataattaa 2108 7 345 PRT Homo sapiens 7 Met Ser Leu Phe Gly Leu Leu Leu Val Thr Ser Ala Leu Ala Gly Gln 1 5 10 15 Arg Arg Gly Thr Gln Ala Glu Ser Asn Leu Ser Ser Lys Phe Gln Phe 20 25 30 Ser Ser Asn Lys Glu Gln Asn Gly Val Gln Asp Pro Gln His Glu Arg 35 40 45 Ile Ile Thr Val Ser Thr Asn Gly Ser Ile His Ser Pro Arg Phe Pro 50 55 60 His Thr Tyr Pro Arg Asn Thr Val Leu Val Trp Arg Leu Val Ala Val 65 70 75 80 Glu Glu Asn Val Trp Ile Gln Leu Thr Phe Asp Glu Arg Phe Gly Leu 85 90 95 Glu Asp Pro Glu Asp Asp Ile Cys Lys Tyr Asp Phe Val Glu Val Glu 100 105 110 Glu Pro Ser Asp Gly Thr Ile Leu Gly Arg Trp Cys Gly Ser Gly Thr 115 120 125 Val Pro Gly Lys Gln Ile Ser Lys Gly Asn Gln Ile Arg Ile Arg Phe 130 135 140 Val Ser Asp Glu Tyr Phe Pro Ser Glu Pro Gly Phe Cys Ile His Tyr 145 150 155 160 Asn Ile Val Met Pro Gln Phe Thr Glu Ala Val Ser Pro Ser Val Leu 165 170 175 Pro Pro Ser Ala Leu Pro Leu Asp Leu Leu Asn Asn Ala Ile Thr Ala 180 185 190 Phe Ser Thr Leu Glu Asp Leu Ile Arg Tyr Leu Glu Pro Glu Arg Trp 195 200 205 Gln Leu Asp Leu Glu Asp Leu Tyr Arg Pro Thr Trp Gln Leu Leu Gly 210 215 220 Lys Ala Phe Val Phe Gly Arg Lys Ser Arg Val Val Asp Leu Asn Leu 225 230 235 240 Leu Thr Glu Glu Val Arg Leu Tyr Ser Cys Thr Pro Arg Asn Phe Ser 245 250 255 Val Ser Ile Arg Glu Glu Leu Lys Arg Thr Asp Thr Ile Phe Trp Pro 260 265 270 Gly Cys Leu Leu Val Lys Arg Cys Gly Gly Asn Cys Ala Cys Cys Leu 275 280 285 His Asn Cys Asn Glu Cys Gln Cys Val Pro Ser Lys Val Thr Lys Lys 290 295 300 Tyr His Glu Val Leu Gln Leu Arg Pro Lys Thr Gly Val Arg Gly Leu 305 310 315 320 His Lys Ser Leu Thr Asp Val Ala Leu Glu His His Glu Glu Cys Asp 325 330 335 Cys Val Cys Arg Gly Ser Thr Gly Gly 340 345 8 2253 DNA Homo sapiens CDS (176)..(1288) 8 cgctcggaaa gttcagcatg caggaagttt ggggagagct cggcgattag cacagcgacc 60 cgggccagcg cagggcgagc gcaggcggcg agagcgcagg gcggcgcggc gtcggtcccg 120 ggagcagaac ccggcttttt cttggagcga cgctgtctct agtcgctgat cccaa atg 178 Met 1 cac cgg ctc atc ttt gtc tac act cta atc tgc gca aac ttt tgc agc 226 His Arg Leu Ile Phe Val Tyr Thr Leu Ile Cys Ala Asn Phe Cys Ser 5 10 15 tgt cgg gac act tct gca acc ccg cag agc gca tcc atc aaa gct ttg 274 Cys Arg Asp Thr Ser Ala Thr Pro Gln Ser Ala Ser Ile Lys Ala Leu 20 25 30 cgc aac gcc aac ctc agg cga gat gag agc aat cac ctc aca gac ttg 322 Arg Asn Ala Asn Leu Arg Arg Asp Glu Ser Asn His Leu Thr Asp Leu 35 40 45 tac cga aga gat gag acc atc cag gtg aaa gga aac ggc tac gtg cag 370 Tyr Arg Arg Asp Glu Thr Ile Gln Val Lys Gly Asn Gly Tyr Val Gln 50 55 60 65 agt cct aga ttc ccg aac agc tac ccc agg aac ctg ctc ctg aca tgg 418 Ser Pro Arg Phe Pro Asn Ser Tyr Pro Arg Asn Leu Leu Leu Thr Trp 70 75 80 cgg ctt cac tct cag gag aat aca cgg ata cag cta gtg ttt gac aat 466 Arg Leu His Ser Gln Glu Asn Thr Arg Ile Gln Leu Val Phe Asp Asn 85 90 95 cag ttt gga tta gag gaa gca gaa aat gat atc tgt agg tat gat ttt 514 Gln Phe Gly Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Tyr Asp Phe 100 105 110 gtg gaa gtt gaa gat ata tcc gaa acc agt acc att att aga gga cga 562 Val Glu Val Glu Asp Ile Ser Glu Thr Ser Thr Ile Ile Arg Gly Arg 115 120 125 tgg tgt gga cac aag gaa gtt cct cca agg ata aaa tca aga acg aac 610 Trp Cys Gly His Lys Glu Val Pro Pro Arg Ile Lys Ser Arg Thr Asn 130 135 140 145 caa att aaa atc aca ttc aag tcc gat gac tac ttt gtg gct aaa cct 658 Gln Ile Lys Ile Thr Phe Lys Ser Asp Asp Tyr Phe Val Ala Lys Pro 150 155 160 gga ttc aag att tat tat tct ttg ctg gaa gat ttc caa ccc gca gca 706 Gly Phe Lys Ile Tyr Tyr Ser Leu Leu Glu Asp Phe Gln Pro Ala Ala 165 170 175 gct tca gag acc aac tgg gaa tct gtc aca agc tct att tca ggg gta 754 Ala Ser Glu Thr Asn Trp Glu Ser Val Thr Ser Ser Ile Ser Gly Val 180 185 190 tcc tat aac tct cca tca gta acg gat ccc act ctg att gcg gat gct 802 Ser Tyr Asn Ser Pro Ser Val Thr Asp Pro Thr Leu Ile Ala Asp Ala 195 200 205 ctg gac aaa aaa att gca gaa ttt gat aca gtg gaa gat ctg ctc aag 850 Leu Asp Lys Lys Ile Ala Glu Phe Asp Thr Val Glu Asp Leu Leu Lys 210 215 220 225 tac ttc aat cca gag tca tgg caa gaa gat ctt gag aat atg tat ctg 898 Tyr Phe Asn Pro Glu Ser Trp Gln Glu Asp Leu Glu Asn Met Tyr Leu 230 235 240 gac acc cct cgg tat cga ggc agg tca tac cat gac cgg aag tca aaa 946 Asp Thr Pro Arg Tyr Arg Gly Arg Ser Tyr His Asp Arg Lys Ser Lys 245 250 255 gtt gac ctg gat agg ctc aat gat gat gcc aag cgt tac agt tgc act 994 Val Asp Leu Asp Arg Leu Asn Asp Asp Ala Lys Arg Tyr Ser Cys Thr 260 265 270 ccc agg aat tac tcg gtc aat ata aga gaa gag ctg aag ttg gcc aat 1042 Pro Arg Asn Tyr Ser Val Asn Ile Arg Glu Glu Leu Lys Leu Ala Asn 275 280 285 gtg gtc ttc ttt cca cgt tgc ctc ctc gtg cag cgc tgt gga gga aat 1090 Val Val Phe Phe Pro Arg Cys Leu Leu Val Gln Arg Cys Gly Gly Asn 290 295 300 305 tgt ggc tgt gga act gtc aac tgg agg tcc tgc aca tgc aat tca ggg 1138 Cys Gly Cys Gly Thr Val Asn Trp Arg Ser Cys Thr Cys Asn Ser Gly 310 315 320 aaa acc gtg aaa aag tat cat gag gta tta cag ttt gag cct ggc cac 1186 Lys Thr Val Lys Lys Tyr His Glu Val Leu Gln Phe Glu Pro Gly His 325 330 335 atc aag agg agg ggt aga gct aag acc atg gct cta gtt gac atc cag 1234 Ile Lys Arg Arg Gly Arg Ala Lys Thr Met Ala Leu Val Asp Ile Gln 340 345 350 ttg gat cac cat gaa cga tgc gat tgt atc tgc agc tca aga cca cct 1282 Leu Asp His His Glu Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro Pro 355 360 365 cga taa gagaatgtgc acatccttac attaagcctg aaagaacctt tagtttaagg 1338 Arg 370 agggtgagat aagagaccct tttcctacca gcaaccaaac ttactactag cctgcaatgc 1398 aatgaacaca agtggttgct gagtctcagc cttgctttgt taatgccatg gcaagtagaa 1458 aggtatatca tcaacttcta tacctaagaa tataggattg catttaataa tagtgtttga 1518 ggttatatat gcacaaacac acacagaaat atattcatgt ctatgtgtat atagatcaaa 1578 tgtttttttt ggtatatata accaggtaca ccagagctta catatgtttg agttagactc 1638 ttaaaatcct ttgccaaaat aagggatggt caaatatatg aaacatgtct ttagaaaatt 1698 taggagataa atttattttt aaattttgaa acacaaaaca attttgaatc ttgctctctt 1758 aaagaaagca tcttgtatat taaaaatcaa aagatgaggc tttcttacat atacatctta 1818 gttgattatt aaaaaaggaa aaaggtttcc agagaaaagg ccaataccta agcatttttt 1878 ccatgagaag cactgcatac ttacctatgt ggactgtaat aacctgtctc caaaaccatg 1938 ccataataat ataagtgctt tagaaattaa atcattgtgt tttttatgca ttttgctgag 1998 gcatccttat tcatttaaca cctatctcaa aaacttactt agaaggtttt ttattatagt 2058 cctacaaaag acaatgtata agctgtaaca gaattttgaa ttgtttttct ttgcaaaacc 2118 cctccacaaa agcaaatcct ttcaagaatg gcatgggcat tctgtatgaa cctttccaga 2178 tggtgttcag tgaaagatgt gggtagttga gaacttaaaa agtgaacatt gaaacatcga 2238 cgtaactgga aaccg 2253 9 370 PRT Homo sapiens 9 Met His Arg Leu Ile Phe Val Tyr Thr Leu Ile Cys Ala Asn Phe Cys 1 5 10 15 Ser Cys Arg Asp Thr Ser Ala Thr Pro Gln Ser Ala Ser Ile Lys Ala 20 25 30 Leu Arg Asn Ala Asn Leu Arg Arg Asp Glu Ser Asn His Leu Thr Asp 35 40 45 Leu Tyr Arg Arg Asp Glu Thr Ile Gln Val Lys Gly Asn Gly Tyr Val 50 55 60 Gln Ser Pro Arg Phe Pro Asn Ser Tyr Pro Arg Asn Leu Leu Leu Thr 65 70 75 80 Trp Arg Leu His Ser Gln Glu Asn Thr Arg Ile Gln Leu Val Phe Asp 85 90 95 Asn Gln Phe Gly Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Tyr Asp 100 105 110 Phe Val Glu Val Glu Asp Ile Ser Glu Thr Ser Thr Ile Ile Arg Gly 115 120 125 Arg Trp Cys Gly His Lys Glu Val Pro Pro Arg Ile Lys Ser Arg Thr 130 135 140 Asn Gln Ile Lys Ile Thr Phe Lys Ser Asp Asp Tyr Phe Val Ala Lys 145 150 155 160 Pro Gly Phe Lys Ile Tyr Tyr Ser Leu Leu Glu Asp Phe Gln Pro Ala 165 170 175 Ala Ala Ser Glu Thr Asn Trp Glu Ser Val Thr Ser Ser Ile Ser Gly 180 185 190 Val Ser Tyr Asn Ser Pro Ser Val Thr Asp Pro Thr Leu Ile Ala Asp 195 200 205 Ala Leu Asp Lys Lys Ile Ala Glu Phe Asp Thr Val Glu Asp Leu Leu 210 215 220 Lys Tyr Phe Asn Pro Glu Ser Trp Gln Glu Asp Leu Glu Asn Met Tyr 225 230 235 240 Leu Asp Thr Pro Arg Tyr Arg Gly Arg Ser Tyr His Asp Arg Lys Ser 245 250 255 Lys Val Asp Leu Asp Arg Leu Asn Asp Asp Ala Lys Arg Tyr Ser Cys 260 265 270 Thr Pro Arg Asn Tyr Ser Val Asn Ile Arg Glu Glu Leu Lys Leu Ala 275 280 285 Asn Val Val Phe Phe Pro Arg Cys Leu Leu Val Gln Arg Cys Gly Gly 290 295 300 Asn Cys Gly Cys Gly Thr Val Asn Trp Arg Ser Cys Thr Cys Asn Ser 305 310 315 320 Gly Lys Thr Val Lys Lys Tyr His Glu Val Leu Gln Phe Glu Pro Gly 325 330 335 His Ile Lys Arg Arg Gly Arg Ala Lys Thr Met Ala Leu Val Asp Ile 340 345 350 Gln Leu Asp His His Glu Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro 355 360 365 Pro Arg 370 10 116 PRT Artificial sequence PDGF-C Core Domain 10 Gly Arg Lys Ser Arg Val Val Asp Leu Asn Leu Leu Thr Glu Glu Val 1 5 10 15 Arg Leu Tyr Ser Cys Thr Pro Arg Asn Phe Ser Val Ser Ile Arg Glu 20 25 30 Glu Leu Lys Arg Thr Asp Thr Ile Phe Trp Pro Gly Cys Leu Leu Val 35 40 45 Lys Arg Cys Gly Gly Asn Cys Ala Cys Cys Leu His Asn Cys Asn Glu 50 55 60 Cys Gln Cys Val Pro Ser Lys Val Thr Lys Lys Tyr His Glu Val Leu 65 70 75 80 Gln Leu Arg Pro Lys Thr Gly Val Arg Gly Leu His Lys Ser Leu Thr 85 90 95 Asp Val Ala Leu Glu His His Glu Glu Cys Asp Cys Val Cys Arg Gly 100 105 110 Ser Thr Gly Gly 115 11 990 DNA Homo sapiens CDS (57)..(629) 11 cagtgtgctg gcggcccggc gcgagccggc ccggccccgg tcgggcctcc gaaacc atg 59 Met 1 aac ttt ctg ctg tct tgg gtg cat tgg agc ctc gcc ttg ctg ctc tac 107 Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu Tyr 5 10 15 ctc cac cat gcc aag tgg tcc cag gct gca ccc atg gca gaa gga gga 155 Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly Gly 20 25 30 ggg cag aat cat cac gaa gtg gtg aag ttc atg gat gtc tat cag cgc 203 Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln Arg 35 40 45 agc tac tgc cat cca atc gag acc ctg gtg gac atc ttc cag gag tac 251 Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr 50 55 60 65 cct gat gag atc gag tac atc ttc aag cca tcc tgt gtg ccc ctg atg 299 Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met 70 75 80 cga tgc ggg ggc tgc tgc aat gac gag ggc ctg gag tgt gtg ccc act 347 Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr 85 90 95 gag gag tcc aac atc acc atg cag att atg cgg atc aaa cct cac caa 395 Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln 100 105 110 ggc cag cac ata gga gag atg agc ttc cta cag cac aac aaa tgt gaa 443 Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu 115 120 125 tgc aga cca aag aaa gat aga gca aga caa gaa aat ccc tgt ggg cct 491 Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly Pro 130 135 140 145 tgc tca gag cgg aga aag cat ttg ttt gta caa gat ccg cag acg tgt 539 Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr Cys 150 155 160 aaa tgt tcc tgc aaa aac aca gac tcg cgt tgc aag gcg agg cag ctt 587 Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu 165 170 175 gag tta aac gaa cgt act tgc aga tgt gac aag ccg agg cgg 629 Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg 180 185 190 tgagccgggc aggaggaagg agcctccctc agggtttcgg gaaccagatc tctcaccagg 689 aaagactgat acagaacgat cgatacagaa accacgctgc cgccaccaca ccatcaccat 749 cgacagaaca gtccttaatc cagaaacctg aaatgaagga agaggagact ctgcgcagag 809 cactttgggt ccggagggcg agactccggc ggaagcattc ccgggcgggt gacccagcac 869 ggtccctctt ggaattggat tcgccatttt atttttcttg ctgctaaatc accgagcccg 929 gaagattaga gagttttatt tctgggattc ctgtagacac accgcggccg ccagcacact 989 g 990 12 191 PRT Homo sapiens 12 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly 130 135 140 Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr 145 150 155 160 Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln 165 170 175 Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg 180 185 190 13 1997 DNA Homo sapiens CDS (352)..(1608) 13 cccgccccgc ctctccaaaa agctacaccg acgcggaccg cggcggcgtc ctccctcgcc 60 ctcgcttcac ctcgcgggct ccgaatgcgg ggagctcgga tgtccggttt cctgtgaggc 120 ttttacctga cacccgccgc ctttccccgg cactggctgg gagggcgccc tgcaaagttg 180 ggaacgcgga gccccggacc cgctcccgcc gcctccggct cgcccagggg gggtcgccgg 240 gaggagcccg ggggagaggg accaggaggg gcccgcggcc tcgcaggggc gcccgcgccc 300 ccacccctgc ccccgccagc ggaccggtcc cccacccccg gtccttccac c atg cac 357 Met His 1 ttg ctg ggc ttc ttc tct gtg gcg tgt tct ctg ctc gcc gct gcg ctg 405 Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala Ala Leu 5 10 15 ctc ccg ggt cct cgc gag gcg ccc gcc gcc gcc gcc gcc ttc gag tcc 453 Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe Glu Ser 20 25 30 gga ctc gac ctc tcg gac gcg gag ccc gac gcg ggc gag gcc acg gct 501 Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala Thr Ala 35 40 45 50 tat gca agc aaa gat ctg gag gag cag tta cgg tct gtg tcc agt gta 549 Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser Ser Val 55 60 65 gat gaa ctc atg act gta ctc tac cca gaa tat tgg aaa atg tac aag 597 Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met Tyr Lys 70 75 80 tgt cag cta agg aaa gga ggc tgg caa cat aac aga gaa cag gcc aac 645 Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln Ala Asn 85 90 95 ctc aac tca agg aca gaa gag act ata aaa ttt gct gca gca cat tat 693 Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala His Tyr 100 105 110 aat aca gag atc ttg aaa agt att gat aat gag tgg aga aag act caa 741 Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr Gln 115 120 125 130 tgc atg cca cgg gag gtg tgt ata gat gtg ggg aag gag ttt gga gtc 789 Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe Gly Val 135 140 145 gcg aca aac acc ttc ttt aaa cct cca tgt gtg tcc gtc tac aga tgt 837 Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr Arg Cys 150 155 160 ggg ggt tgc tgc aat agt gag ggg ctg cag tgc atg aac acc agc acg 885 Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr Ser Thr 165 170 175 agc tac ctc agc aag acg tta ttt gaa att aca gtg cct ctc tct caa 933 Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu Ser Gln 180 185 190 ggc ccc aaa cca gta aca atc agt ttt gcc aat cac act tcc tgc cga 981 Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser Cys Arg 195 200 205 210 tgc atg tct aaa ctg gat gtt tac aga caa gtt cat tcc att att aga 1029 Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile Ile Arg 215 220 225 cgt tcc ctg cca gca aca cta cca cag tgt cag gca gcg aac aag acc 1077 Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn Lys Thr 230 235 240 tgc ccc acc aat tac atg tgg aat aat cac atc tgc aga tgc ctg gct 1125 Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys Leu Ala 245 250 255 cag gaa gat ttt atg ttt tcc tcg gat gct gga gat gac tca aca gat 1173 Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser Thr Asp 260 265 270 gga ttc cat gac atc tgt gga cca aac aag gag ctg gat gaa gag acc 1221 Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu Glu Thr 275 280 285 290 tgt cag tgt gtc tgc aga gcg ggg ctt cgg cct gcc agc tgt gga ccc 1269 Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys Gly Pro 295 300 305 cac aaa gaa cta gac aga aac tca tgc cag tgt gtc tgt aaa aac aaa 1317 His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys 310 315 320 ctc ttc ccc agc caa tgt ggg gcc aac cga gaa ttt gat gaa aac aca 1365 Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu Asn Thr 325 330 335 tgc cag tgt gta tgt aaa aga acc tgc ccc aga aat caa ccc cta aat 1413 Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn 340 345 350 cct gga aaa tgt gcc tgt gaa tgt aca gaa agt cca cag aaa tgc ttg 1461 Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys Cys Leu 355 360 365 370 tta aaa gga aag aag ttc cac cac caa aca tgc agc tgt tac aga cgg 1509 Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr Arg Arg 375 380 385 cca tgt acg aac cgc cag aag gct tgt gag cca gga ttt tca tat agt 1557 Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser Tyr Ser 390 395 400 gaa gaa gtg tgt cgt tgt gtc cct tca tat tgg aaa aga cca caa atg 1605 Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro Gln Met 405 410 415 agc taagattgta ctgttttcca gttcatcgat tttctattat ggaaaactgt 1658 Ser gttgccacag tagaactgtc tgtgaacaga gagacccttg tgggtccatg ctaacaaaga 1718 caaaagtctg tctttcctga accatgtgga taactttaca gaaatggact ggagctcatc 1778 tgcaaaaggc ctcttgtaaa gactggtttt ctgccaatga ccaaacagcc aagattttcc 1838 tcttgtgatt tctttaaaag aatgactata taatttattt ccactaaaaa tattgtttct 1898 gcattcattt ttatagcaac aacaattggt aaaactcact gtgatcaata tttttatatc 1958 atgcaaaata tgtttaaaat aaaatgaaaa ttgtattat 1997 14 419 PRT Homo sapiens 14 Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala 1 5 10 15 Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe 20 25 30 Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala 35 40 45 Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser 50 55 60 Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr Trp Lys Met 65 70 75 80 Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln 85 90 95 Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala 100 105 110 His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys 115 120 125 Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe 130 135 140 Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val Tyr 145 150 155 160 Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met Asn Thr 165 170 175 Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu 180 185 190 Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser 195 200 205 Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile 210 215 220 Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn 225 230 235 240 Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys 245 250 255 Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser 260 265 270 Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu 275 280 285 Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys 290 295 300 Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys 305 310 315 320 Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu Phe Asp Glu 325 330 335 Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro 340 345 350 Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys 355 360 365 Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr 370 375 380 Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly Phe Ser 385 390 395 400 Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp Lys Arg Pro 405 410 415 Gln Met Ser 15 1645 DNA Homo sapiens CDS (322)..(768) 15 gggattcggg ccgcccagct acgggaggac ctggagtggc actgggcgcc cgacggacca 60 tccccgggac ccgcctgccc ctcggcgccc cgccccgccg ggccgctccc cgtcgggttc 120 cccagccaca gccttaccta cgggctcctg actccgcaag gcttccagaa gatgctcgaa 180 ccaccggccg gggcctcggg gcagcagtga gggaggcgtc cagcccccca ctcagctctt 240 ctcctcctgt gccaggggct ccccggggga tgagcatggt ggttttccct cggagccccc 300 tggctcggga cgtctgagaa g atg ccg gtc atg agg ctg ttc cct tgc ttc 351 Met Pro Val Met Arg Leu Phe Pro Cys Phe 1 5 10 ctg cag ctc ctg gcc ggg ctg gcg ctg cct gct gtg ccc ccc cag cag 399 Leu Gln Leu Leu Ala Gly Leu Ala Leu Pro Ala Val Pro Pro Gln Gln 15 20 25 tgg gcc ttg tct gct ggg aac ggc tcg tca gag gtg gaa gtg gta ccc 447 Trp Ala Leu Ser Ala Gly Asn Gly Ser Ser Glu Val Glu Val Val Pro 30 35 40 ttc cag gaa gtg tgg ggc cgc agc tac tgc cgg gcg ctg gag agg ctg 495 Phe Gln Glu Val Trp Gly Arg Ser Tyr Cys Arg Ala Leu Glu Arg Leu 45 50 55 gtg gac gtc gtg tcc gag tac ccc agc gag gtg gag cac atg ttc agc 543 Val Asp Val Val Ser Glu Tyr Pro Ser Glu Val Glu His Met Phe Ser 60 65 70 cca tcc tgt gtc tcc ctg ctg cgc tgc acc ggc tgc tgc ggc gat gag 591 Pro Ser Cys Val Ser Leu Leu Arg Cys Thr Gly Cys Cys Gly Asp Glu 75 80 85 90 aat ctg cac tgt gtg ccg gtg gag acg gcc aat gtc acc atg cag ctc 639 Asn Leu His Cys Val Pro Val Glu Thr Ala Asn Val Thr Met Gln Leu 95 100 105 cta aag atc cgt tct ggg gac cgg ccc tcc tac gtg gag ctg acg ttc 687 Leu Lys Ile Arg Ser Gly Asp Arg Pro Ser Tyr Val Glu Leu Thr Phe 110 115 120 tct cag cac gtt cgc tgc gaa tgc cgg cct ctg cgg gag aag atg aag 735 Ser Gln His Val Arg Cys Glu Cys Arg Pro Leu Arg Glu Lys Met Lys 125 130 135 ccg gaa agg tgc ggc gat gct gtt ccc cgg agg taacccaccc cttggaggag 788 Pro Glu Arg Cys Gly Asp Ala Val Pro Arg Arg 140 145 agagaccccg cacccggctc gtgtatttat taccgtcaca ctcttcagtg actcctgctg 848 gtacctgccc tctatttatt agccaactgt ttccctgctg aatgcctcgc tcccttcaag 908 acgaggggca gggaaggaca ggaccctcag gaattcagtg ccttcaacaa cgtgagagaa 968 agagagaagc cagccacaga cccctgggag cttccgcttt gaaagaagca agacacgtgg 1028 cctcgtgagg ggcaagctag gccccagagg ccctggaggt ctccaggggc ctgcagaagg 1088 aaagaagggg gccctgctac ctgttcttgg gcctcaggct ctgcacagac aagcagccct 1148 tgctttcgga gctcctgtcc aaagtaggga tgcggattct gctggggccg ccacggcctg 1208 gtggtgggaa ggccggcagc gggcggaggg gattcagcca cttccccctc ttcttctgaa 1268 gatcagaaca ttcagctctg gagaacagtg gttgcctggg ggcttttgcc actccttgtc 1328 ccccgtgatc tcccctcaca ctttgccatt tgcttgtact gggacattgt tctttccggc 1388 cgaggtgcca ccaccctgcc cccactaaga gacacataca gagtgggccc cgggctggag 1448 aaagagctgc ctggatgaga aacagctcag ccagtgggga tgaggtcacc aggggaggag 1508 cctgtgcgtc ccagctgaag gcagtggcag gggagcaggt tccccaaggg ccctggcacc 1568 cccacaagct gtccctgcag ggccatctga ctgccaagcc agattctctt gaataaagta 1628 ttctagtgtg gaaacgc 1645 16 149 PRT Homo sapiens 16 Met Pro Val Met Arg Leu Phe Pro Cys Phe Leu Gln Leu Leu Ala Gly 1 5 10 15 Leu Ala Leu Pro Ala Val Pro Pro Gln Gln Trp Ala Leu Ser Ala Gly 20 25 30 Asn Gly Ser Ser Glu Val Glu Val Val Pro Phe Gln Glu Val Trp Gly 35 40 45 Arg Ser Tyr Cys Arg Ala Leu Glu Arg Leu Val Asp Val Val Ser Glu 50 55 60 Tyr Pro Ser Glu Val Glu His Met Phe Ser Pro Ser Cys Val Ser Leu 65 70 75 80 Leu Arg Cys Thr Gly Cys Cys Gly Asp Glu Asn Leu His Cys Val Pro 85 90 95 Val Glu Thr Ala Asn Val Thr Met Gln Leu Leu Lys Ile Arg Ser Gly 100 105 110 Asp Arg Pro Ser Tyr Val Glu Leu Thr Phe Ser Gln His Val Arg Cys 115 120 125 Glu Cys Arg Pro Leu Arg Glu Lys Met Lys Pro Glu Arg Cys Gly Asp 130 135 140 Ala Val Pro Arg Arg 145 17 2029 DNA Homo sapiens CDS (411)..(1472) 17 gttgggttcc agctttctgt agctgtaagc attggtggcc acaccacctc cttacaaagc 60 aactagaacc tgcggcatac attggagaga tttttttaat tttctggaca tgaagtaaat 120 ttagagtgct ttctaatttc aggtagaaga catgtccacc ttctgattat ttttggagaa 180 cattttgatt tttttcatct ctctctcccc acccctaaga ttgtgcaaaa aaagcgtacc 240 ttgcctaatt gaaataattt cattggattt tgatcagaac tgattatttg gttttctgtg 300 tgaagttttg aggtttcaaa ctttccttct ggagaatgcc ttttgaaaca attttctcta 360 gctgcctgat gtcaactgct tagtaatcag tggatattga aatattcaaa atg tac 416 Met Tyr 1 aga gag tgg gta gtg gtg aat gtt ttc atg atg ttg tac gtc cag ctg 464 Arg Glu Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val Gln Leu 5 10 15 gtg cag ggc tcc agt aat gaa cat gga cca gtg aag cga tca tct cag 512 Val Gln Gly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser Ser Gln 20 25 30 tcc aca ttg gaa cga tct gaa cag cag atc agg gct gct tct agt ttg 560 Ser Thr Leu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser Ser Leu 35 40 45 50 gag gaa cta ctt cga att act cac tct gag gac tgg aag ctg tgg aga 608 Glu Glu Leu Leu Arg Ile Thr His Ser Glu Asp Trp Lys Leu Trp Arg 55 60 65 tgc agg ctg agg ctc aaa agt ttt acc agt atg gac tct cgc tca gca 656 Cys Arg Leu Arg Leu Lys Ser Phe Thr Ser Met Asp Ser Arg Ser Ala 70 75 80 tcc cat cgg tcc act agg ttt gcg gca act ttc tat gac att gaa aca 704 Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Ile Glu Thr 85 90 95 cta aaa gtt ata gat gaa gaa tgg caa aga act cag tgc agc cct aga 752 Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser Pro Arg 100 105 110 gaa acg tgc gtg gag gtg gcc agt gag ctg ggg aag agt acc aac aca 800 Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr Asn Thr 115 120 125 130 ttc ttc aag ccc cct tgt gtg aac gtg ttc cga tgt ggt ggc tgt tgc 848 Phe Phe Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly Cys Cys 135 140 145 aat gaa gag agc ctt atc tgt atg aac acc agc acc tcg tac att tcc 896 Asn Glu Glu Ser Leu Ile Cys Met Asn Thr Ser Thr Ser Tyr Ile Ser 150 155 160 aaa cag ctc ttt gag ata tca gtg cct ttg aca tca gta cct gaa tta 944 Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro Glu Leu 165 170 175 gtg cct gtt aaa gtt gcc aat cat aca ggt tgt aag tgc ttg cca aca 992 Val Pro Val Lys Val Ala Asn His Thr Gly Cys Lys Cys Leu Pro Thr 180 185 190 gcc ccc cgc cat cca tac tca att atc aga aga tcc atc cag atc cct 1040 Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln Ile Pro 195 200 205 210 gaa gaa gat cgc tgt tcc cat tcc aag aaa ctc tgt cct att gac atg 1088 Glu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile Asp Met 215 220 225 cta tgg gat agc aac aaa tgt aaa tgt gtt ttg cag gag gaa aat cca 1136 Leu Trp Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu Asn Pro 230 235 240 ctt gct gga aca gaa gac cac tct cat ctc cag gaa cca gct ctc tgt 1184 Leu Ala Gly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala Leu Cys 245 250 255 ggg cca cac atg atg ttt gac gaa gat cgt tgc gag tgt gtc tgt aaa 1232 Gly Pro His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val Cys Lys 260 265 270 aca cca tgt ccc aaa gat cta atc cag cac ccc aaa aac tgc agt tgc 1280 Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro Lys Asn Cys Ser Cys 275 280 285 290 ttt gag tgc aaa gaa agt ctg gag acc tgc tgc cag aag cac aag cta 1328 Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His Lys Leu 295 300 305 ttt cac cca gac acc tgc agc tgt gag gac aga tgc ccc ttt cat acc 1376 Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe His Thr 310 315 320 aga cca tgt gca agt ggc aaa aca gca tgt gca aag cat tgc cgc ttt 1424 Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys Arg Phe 325 330 335 cca aag gag aaa agg gct gcc cag ggg ccc cac agc cga aag aat cct 1472 Pro Lys Glu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys Asn Pro 340 345 350 tgattcagcg ttccaagttc cccatccctg tcatttttaa cagcatgctg ctttgccaag 1532 ttgctgtcac tgtttttttc ccaggtgtta aaaaaaaaat ccattttaca cagcaccaca 1592 gtgaatccag accaaccttc cattcacacc agctaaggag tccctggttc attgatggat 1652 gtcttctagc tgcagatgcc tctgcgcacc aaggaatgga gaggagggga cccatgtaat 1712 ccttttgttt agttttgttt ttgttttttg gtgaatgaga aaggtgtgct ggtcatggaa 1772 tggcaggtgt catatgactg attactcaga gcagatgagg aaaactgtag tctctgagtc 1832 ctttgctaat cgcaactctt gtgaattatt ctgattcttt tttatgcaga atttgattcg 1892 tatgatcagt actgactttc tgattactgt ccagcttata gtcttccagt ttaatgaact 1952 accatctgat gtttcatatt taagtgtatt taaagaaaat aaacaccatt attcaagcca 2012 aaaaaaaaaa aaaaaaa 2029 18 354 PRT Homo sapiens 18 Met Tyr Arg Glu Trp Val Val Val Asn Val Phe Met Met Leu Tyr Val 1 5 10 15 Gln Leu Val Gln Gly Ser Ser Asn Glu His Gly Pro Val Lys Arg Ser 20 25 30 Ser Gln Ser Thr Leu Glu Arg Ser Glu Gln Gln Ile Arg Ala Ala Ser 35 40 45 Ser Leu Glu Glu Leu Leu Arg Ile Thr His Ser Glu Asp Trp Lys Leu 50 55 60 Trp Arg Cys Arg Leu Arg Leu Lys Ser Phe Thr Ser Met Asp Ser Arg 65 70 75 80 Ser Ala Ser His Arg Ser Thr Arg Phe Ala Ala Thr Phe Tyr Asp Ile 85 90 95 Glu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln Arg Thr Gln Cys Ser 100 105 110 Pro Arg Glu Thr Cys Val Glu Val Ala Ser Glu Leu Gly Lys Ser Thr 115 120 125 Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val Phe Arg Cys Gly Gly 130 135 140 Cys Cys Asn Glu Glu Ser Leu Ile Cys Met Asn Thr Ser Thr Ser Tyr 145 150 155 160 Ile Ser Lys Gln Leu Phe Glu Ile Ser Val Pro Leu Thr Ser Val Pro 165 170 175 Glu Leu Val Pro Val Lys Val Ala Asn His Thr Gly Cys Lys Cys Leu 180 185 190 Pro Thr Ala Pro Arg His Pro Tyr Ser Ile Ile Arg Arg Ser Ile Gln 195 200 205 Ile Pro Glu Glu Asp Arg Cys Ser His Ser Lys Lys Leu Cys Pro Ile 210 215 220 Asp Met Leu Trp Asp Ser Asn Lys Cys Lys Cys Val Leu Gln Glu Glu 225 230 235 240 Asn Pro Leu Ala Gly Thr Glu Asp His Ser His Leu Gln Glu Pro Ala 245 250 255 Leu Cys Gly Pro His Met Met Phe Asp Glu Asp Arg Cys Glu Cys Val 260 265 270 Cys Lys Thr Pro Cys Pro Lys Asp Leu Ile Gln His Pro Lys Asn Cys 275 280 285 Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Thr Cys Cys Gln Lys His 290 295 300 Lys Leu Phe His Pro Asp Thr Cys Ser Cys Glu Asp Arg Cys Pro Phe 305 310 315 320 His Thr Arg Pro Cys Ala Ser Gly Lys Thr Ala Cys Ala Lys His Cys 325 330 335 Arg Phe Pro Lys Glu Lys Arg Ala Ala Gln Gly Pro His Ser Arg Lys 340 345 350 Asn Pro 19 1830 DNA Orf virus CDS (312)..(755) 19 cggccacgcg gccgcgaact gcgcgctcgc gcgcgtggcg accgcgctga cgcgccgcgt 60 gcccgcgagc cggcacggcc tcgcggaggg cggcacgccg ccgtggacgc tgctgctggc 120 ggtggccgcg gtggcggtgc tcggcgtggt ggcaatttcg ctgctgcgcc gcgcgctaag 180 aatacggttt agatactcaa agtctatcca gacacttaga gtgtaacttt gagtaaaaaa 240 tgtaaatact aacgccaaaa tttcgatagt tgttaagcaa tatataacat ttttaaaacg 300 tcatcaccag c atg aag tta aca gct acg tta caa gtt gtt gtt gca ttg 350 Met Lys Leu Thr Ala Thr Leu Gln Val Val Val Ala Leu 1 5 10 tta ata tgt atg tat aat ttg cca gaa tgc gtg tct cag agt aat gat 398 Leu Ile Cys Met Tyr Asn Leu Pro Glu Cys Val Ser Gln Ser Asn Asp 15 20 25 tca cct cct tca acc aat gac tgg atg cgt aca cta gac aaa agt ggt 446 Ser Pro Pro Ser Thr Asn Asp Trp Met Arg Thr Leu Asp Lys Ser Gly 30 35 40 45 tgt aaa cct aga gat act gtt gtt tat ttg gga gaa gaa tat cca gaa 494 Cys Lys Pro Arg Asp Thr Val Val Tyr Leu Gly Glu Glu Tyr Pro Glu 50 55 60 agc act aac cta caa tat aat ccc cgg tgc gta act gtt aaa cga tgc 542 Ser Thr Asn Leu Gln Tyr Asn Pro Arg Cys Val Thr Val Lys Arg Cys 65 70 75 agt ggt tgc tgt aac ggt gac ggt caa ata tgt aca gcg gtt gaa aca 590 Ser Gly Cys Cys Asn Gly Asp Gly Gln Ile Cys Thr Ala Val Glu Thr 80 85 90 aga aat aca act gta aca gtt tca gta acc ggc gtg tct agt tcg tct 638 Arg Asn Thr Thr Val Thr Val Ser Val Thr Gly Val Ser Ser Ser Ser 95 100 105 ggt act aat agt ggt gta tct act aac ctt caa aga ata agt gtt aca 686 Gly Thr Asn Ser Gly Val Ser Thr Asn Leu Gln Arg Ile Ser Val Thr 110 115 120 125 gaa cac aca aag tgc gat tgt att ggt aga aca acg aca aca cct acg 734 Glu His Thr Lys Cys Asp Cys Ile Gly Arg Thr Thr Thr Thr Pro Thr 130 135 140 acc act agg gaa cct aga cga taactaataa caaaaaatgt ttatttttgt 785 Thr Thr Arg Glu Pro Arg Arg 145 aaatacttaa ttattacaca ctttacaata atctcaaaaa taaattgcgt gcccggacgg 845 ctgcagctgg tgacgctgct gtgtcacaca ctgcgtattc gattcaagtt cactaacgcc 905 actaaactag ttgtgcgtgt ccgagtgtta accgtacgtc aaactaacat cttacctgtc 965 cgtgacaaga actaaaactt gaaccacata tttttaaagt atatttaaca aaatcactca 1025 cactcacaca atcataaaca ccacaaccac aaccaaacac gcatgagaat taatattctt 1085 acttatccgt aacactctat gctgtacatc aacgcatcag agcagtctga gtctgactaa 1145 tggcggcaaa cgggaacgca ggcgcgacat aatcactgag aatctccgca gcaaccgctc 1205 aaggacatct ctagcgctaa cggctgtttg tcattccccc gtgtgttcat ctcacacgac 1265 attgtgaccg tcgcaaagca cacattcaaa gtgccgcatg tggaagaatt caccgtcgag 1325 acacacacca taattaaaca agatcagtgc ataagagaga ttagcattct acagcacacc 1385 acgtgcgaat acggacctcg taattgttta gactagaaca cctctggtct aaacaacatg 1445 tccgatctta gaacagagtt tatgacgcat atgtaactgt gttctttatg tagaagttat 1505 cttttatgtc actcccttgt cttagatgag ttatacatga catgatgtat gtgtcgcccg 1565 cggcggcgcg gggcgctcgg cggcggggct gctgcgcgcg gcgggcccgc ggtggcggcg 1625 gctggcgcgg cgctgcggcc gcgggcgcgc ggcggggtag cggcccgccc gcccgggcgc 1685 ccgccgcagc ccttgccccg gaccaggcgc cacggagcaa agtgaaaaag gaccgcctag 1745 cagtcgagac cctcccgccg cagccgcgac accccacacc cgccttccac ccgccagacg 1805 ccaacaccac agccaacaag catgc 1830 20 148 PRT Orf virus 20 Met Lys Leu Thr Ala Thr Leu Gln Val Val Val Ala Leu Leu Ile Cys 1 5 10 15 Met Tyr Asn Leu Pro Glu Cys Val Ser Gln Ser Asn Asp Ser Pro Pro 20 25 30 Ser Thr Asn Asp Trp Met Arg Thr Leu Asp Lys Ser Gly Cys Lys Pro 35 40 45 Arg Asp Thr Val Val Tyr Leu Gly Glu Glu Tyr Pro Glu Ser Thr Asn 50 55 60 Leu Gln Tyr Asn Pro Arg Cys Val Thr Val Lys Arg Cys Ser Gly Cys 65 70 75 80 Cys Asn Gly Asp Gly Gln Ile Cys Thr Ala Val Glu Thr Arg Asn Thr 85 90 95 Thr Val Thr Val Ser Val Thr Gly Val Ser Ser Ser Ser Gly Thr Asn 100 105 110 Ser Gly Val Ser Thr Asn Leu Gln Arg Ile Ser Val Thr Glu His Thr 115 120 125 Lys Cys Asp Cys Ile Gly Arg Thr Thr Thr Thr Pro Thr Thr Thr Arg 130 135 140 Glu Pro Arg Arg 145 21 851 DNA Orf virus CDS (2)..(223) 21 c ggc cac gcg gcc gcg aac tgc gcg ctc gcg cgc gtg gcg acc gcg ctg 49 Gly His Ala Ala Ala Asn Cys Ala Leu Ala Arg Val Ala Thr Ala Leu 1 5 10 15 acg cgc cgc gtg ccc gcg agc cgg cac ggc ctc gcg gag ggc ggc acg 97 Thr Arg Arg Val Pro Ala Ser Arg His Gly Leu Ala Glu Gly Gly Thr 20 25 30 ccg ccg tgg acg ctg ctg ctg gcg gtg gcc gcg gtg acg gtg ctc ggc 145 Pro Pro Trp Thr Leu Leu Leu Ala Val Ala Ala Val Thr Val Leu Gly 35 40 45 gtg gtg gcg gtt tca ctg ctg cgg cgc gcg ctg cgg gta cgc tac cgc 193 Val Val Ala Val Ser Leu Leu Arg Arg Ala Leu Arg Val Arg Tyr Arg 50 55 60 ttc gcg cgg ccg gcc gcg ctg cgc gcg tag ccgcgcaaaa tgtaaattat 243 Phe Ala Arg Pro Ala Ala Leu Arg Ala 65 70 aacgcccaac ttttaagggt gaggcgccat gaagttgctc gtcggcatac tagtagccgt 303 gtgcttgcac cagtatctgc tgaacgcgga cagcaacacg aaaggatggt ccgaagtgct 363 gaaaggcagc gagtgcaagc ctaggccgat tgttgttcct gtaagcgaga cgcacccaga 423 gctgacttct cagcggttca acccgccgtg tgtcacgttg atgcgatgcg gcgggtgctg 483 caacgacgag agcttggaat gcgtccccac ggaagaagta aacgtgagca tggaactcct 543 gggggcgtcg ggctccggta gtaacgggat gcaacgtctg agcttcgtag agcataagaa 603 atgcgattgt agaccacgat tcacaaccac gccaccgacg accacaaggc cgcccagaag 663 acgccgctag aactttttat ggaccgcaga tccaaacgat ggatgcgatc aggtacatgc 723 ggaagaaggc gccacggagc aaagtgaaaa aggaccgcct agcagtcgag accctcccgc 783 cgcagccgcg gacaccccac acccgccttc cacccgccag acgccaacac cgcagccaac 843 aagcatgc 851 22 73 PRT Orf virus 22 Gly His Ala Ala Ala Asn Cys Ala Leu Ala Arg Val Ala Thr Ala Leu 1 5 10 15 Thr Arg Arg Val Pro Ala Ser Arg His Gly Leu Ala Glu Gly Gly Thr 20 25 30 Pro Pro Trp Thr Leu Leu Leu Ala Val Ala Ala Val Thr Val Leu Gly 35 40 45 Val Val Ala Val Ser Leu Leu Arg Arg Ala Leu Arg Val Arg Tyr Arg 50 55 60 Phe Ala Arg Pro Ala Ala Leu Arg Ala 65 70 23 2305 DNA Homo sapiens CDS (404)..(991) 23 ttcttggggc tgatgtccgc aaatatgcag aattaccggc cgggtcgctc ctgaagccag 60 cgcggggagc gagcgcggcg gcggccagca ccgggaacgc accgaggaag aagcccagcc 120 cccgccctcc gccccttccg tccccacccc ctacccggcg gcccaggagg ctccccggct 180 gcggcgcgca ctccctgttt ctcctcctcc tggctggcgc tgcctgcctc tccgcactca 240 ctgctcgccg ggcgccgtcc gccagctccg tgctccccgc gccaccctcc tccgggccgc 300 gctccctaag ggatggtact gaatttcgcc gccacaggag accggctgga gcgcccgccc 360 cgcgcctcgc ctctcctccg agcagccagc gcctcgggac gcg atg agg acc ttg 415 Met Arg Thr Leu 1 gct tgc ctg ctg ctc ctc ggc tgc gga tac ctc gcc cat gtt ctg gcc 463 Ala Cys Leu Leu Leu Leu Gly Cys Gly Tyr Leu Ala His Val Leu Ala 5 10 15 20 gag gaa gcc gag atc ccc cgc gag gtg atc gag agg ctg gcc cgc agt 511 Glu Glu Ala Glu Ile Pro Arg Glu Val Ile Glu Arg Leu Ala Arg Ser 25 30 35 cag atc cac agc atc cgg gac ctc cag cga ctc ctg gag ata gac tcc 559 Gln Ile His Ser Ile Arg Asp Leu Gln Arg Leu Leu Glu Ile Asp Ser 40 45 50 gta ggg agt gag gat tct ttg gac acc agc ctg aga gct cac ggg gtc 607 Val Gly Ser Glu Asp Ser Leu Asp Thr Ser Leu Arg Ala His Gly Val 55 60 65 cac gcc act aag cat gtg ccc gag aag cgg ccc ctg ccc att cgg agg 655 His Ala Thr Lys His Val Pro Glu Lys Arg Pro Leu Pro Ile Arg Arg 70 75 80 aag aga agc atc gag gaa gct gtc ccc gct gtc tgc aag acc agg acg 703 Lys Arg Ser Ile Glu Glu Ala Val Pro Ala Val Cys Lys Thr Arg Thr 85 90 95 100 gtc att tac gag att cct cgg agt cag gtc gac ccc acg tcc gcc aac 751 Val Ile Tyr Glu Ile Pro Arg Ser Gln Val Asp Pro Thr Ser Ala Asn 105 110 115 ttc ctg atc tgg ccc ccg tgc gtg gag gtg aaa cgc tgc acc ggc tgc 799 Phe Leu Ile Trp Pro Pro Cys Val Glu Val Lys Arg Cys Thr Gly Cys 120 125 130 tgc aac acg agc agt gtc aag tgc cag ccc tcc cgc gtc cac cac cgc 847 Cys Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg Val His His Arg 135 140 145 agc gtc aag gtg gcc aag gtg gaa tac gtc agg aag aag cca aaa tta 895 Ser Val Lys Val Ala Lys Val Glu Tyr Val Arg Lys Lys Pro Lys Leu 150 155 160 aaa gaa gtc cag gtg agg tta gag gag cat ttg gag tgc gcc tgc gcg 943 Lys Glu Val Gln Val Arg Leu Glu Glu His Leu Glu Cys Ala Cys Ala 165 170 175 180 acc aca agc ctg aat ccg gat tat cgg gaa gag gac acg gat gtg agg 991 Thr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp Thr Asp Val Arg 185 190 195 tgaggatgag ccgcagccct ttcctgggac atggatgtac atggcgtgtt acattcctga 1051 acctactatg tacggtgctt tattgccagt gtgcggtctt tgttctcctc cgtgaaaaac 1111 tgtgtccgag aacactcggg agaacaaaga gacagtgcac atttgtttaa tgtgacatca 1171 aagcaagtat tgtagcactc ggtgaagcag taagaagctt ccttgtcaaa aagagagaga 1231 gagagagaga gagagaaaac aaaaccacaa atgacaaaaa caaaacggac tcacaaaaat 1291 atctaaactc gatgagatgg agggtcgccc cgtgggatgg aagtgcagag gtctcagcag 1351 actggatttc tgtccgggtg gtcacaggtg cttttttgcc gaggatgcag agcctgcttt 1411 gggaacgact ccagaggggt gctggtgggc tctgcagggc ccgcaggaag caggaatgtc 1471 ttggaaaccg ccacgcgaac tttagaaacc acacctcctc gctgtagtat ttaagcccat 1531 acagaaacct tcctgagagc cttaagtggt tttttttttt gtttttgttt tgtttttttt 1591 ttttttgttt tttttttttt tttttttttt tacaccataa agtgattatt aagcttcctt 1651 ttactctttg gctagctttt tttttttttt tttttttttt tttttttaat tatctcttgg 1711 atgacattta caccgataac acacaggctg ctgtaactgt caggacagtg cgacggtatt 1771 tttcctagca agatgcaaac taatgagatg tattaaaata aacatggtat acctacctat 1831 gcatcatttc ctaaatgttt ctggctttgt gtttctccct taccctgctt tatttgttaa 1891 tttaagccat tttgaaagaa ctatgcgtca accaatcgta cgccgtccct gcggcacctg 1951 ccccagagcc cgtttgtggc tgagtgacaa cttgttcccc gcagtgcaca cctagaatgc 2011 tgtgttccca cgcggcacgt gagatgcatt gccgcttctg tctgtgttgt tggtgtgccc 2071 tggtgccgtg gtggcggtca ctccctctgc tgccagtgtt tggacagaac ccaaattctt 2131 tatttttggt aagatattgt gctttacctg tattaacaga aatgtgtgtg tgtggtttgt 2191 ttttttgtaa aggtgaagtt tgtatgttta cctaatatta cctgttttgt atacctgaga 2251 gcctgctatg ttcttctttt gttgatccaa aattaaaaaa aaaataccac caac 2305 24 196 PRT Homo sapiens 24 Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly Cys Gly Tyr Leu Ala 1 5 10 15 His Val Leu Ala Glu Glu Ala Glu Ile Pro Arg Glu Val Ile Glu Arg 20 25 30 Leu Ala Arg Ser Gln Ile His Ser Ile Arg Asp Leu Gln Arg Leu Leu 35 40 45 Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Leu Asp Thr Ser Leu Arg 50 55 60 Ala His Gly Val His Ala Thr Lys His Val Pro Glu Lys Arg Pro Leu 65 70 75 80 Pro Ile Arg Arg Lys Arg Ser Ile Glu Glu Ala Val Pro Ala Val Cys 85 90 95 Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser Gln Val Asp Pro 100 105 110 Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val Glu Val Lys Arg 115 120 125 Cys Thr Gly Cys Cys Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg 130 135 140 Val His His Arg Ser Val Lys Val Ala Lys Val Glu Tyr Val Arg Lys 145 150 155 160 Lys Pro Lys Leu Lys Glu Val Gln Val Arg Leu Glu Glu His Leu Glu 165 170 175 Cys Ala Cys Ala Thr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp 180 185 190 Thr Asp Val Arg 195 25 2137 DNA Homo sapiens CDS (983)..(1705) 25 ccctgcctgc ctccctgcgc acccgcagcc tcccccgctg cctccctagg gctcccctcc 60 ggccgccagc gcccattttt cattccctag atagagatac tttgcgcgca cacacataca 120 tacgcgcgca aaaaggaaaa aaaaaaaaaa aagcccaccc tccagcctcg ctgcaaagag 180 aaaaccggag cagccgcagc tcgcagctcg cagcccgcag cccgcagagg acgcccagag 240 cggcgagcgg gcgggcagac ggaccgacgg actcgcgccg cgtccacctg tcggccgggc 300 ccagccgagc gcgcagcggg cacgccgcgc gcgcggagca gccgtgcccg ccgcccgggc 360 ccgccgccag ggcgcacacg ctcccgcccc cctacccggc ccgggcggga gtttgcacct 420 ctccctgccc gggtgctcga gctgccgttg caaagccaac tttggaaaaa gttttttggg 480 ggagacttgg gccttgaggt gcccagctcc gcgctttccg attttggggg cctttccaga 540 aaatgttgca aaaaagctaa gccggcgggc agaggaaaac gcctgtagcc ggcgagtgaa 600 gacgaaccat cgactgccgt gttccttttc ctcttggagg ttggagtccc ctgggcgccc 660 ccacacggct agacgcctcg gctggttcgc gacgcagccc cccggccgtg gatgctgcac 720 tcgggctcgg gatccgccca ggtagcggcc tcggacccag gtcctgcgcc caggtcctcc 780 cctgcccccc agcgacggag ccggggccgg gggcggcggc gccgggggca tgcgggtgag 840 ccgcggctgc agaggcctga gcgcctgatc gccgcggacc cgagccgagc ccacccccct 900 ccccagcccc ccaccctggc cgcgggggcg gcgcgctcga tctacgcgtt cggggccccg 960 cggggccggg cccggagtcg gc atg aat cgc tgc tgg gcg ctc ttc ctg tct 1012 Met Asn Arg Cys Trp Ala Leu Phe Leu Ser 1 5 10 ctc tgc tgc tac ctg cgt ctg gtc agc gcc gag ggg gac ccc att ccc 1060 Leu Cys Cys Tyr Leu Arg Leu Val Ser Ala Glu Gly Asp Pro Ile Pro 15 20 25 gag gag ctt tat gag atg ctg agt gac cac tcg atc cgc tcc ttt gat 1108 Glu Glu Leu Tyr Glu Met Leu Ser Asp His Ser Ile Arg Ser Phe Asp 30 35 40 gat ctc caa cgc ctg ctg cac gga gac ccc gga gag gaa gat ggg gcc 1156 Asp Leu Gln Arg Leu Leu His Gly Asp Pro Gly Glu Glu Asp Gly Ala 45 50 55 gag ttg gac ctg aac atg acc cgc tcc cac tct gga ggc gag ctg gag 1204 Glu Leu Asp Leu Asn Met Thr Arg Ser His Ser Gly Gly Glu Leu Glu 60 65 70 agc ttg gct cgt gga aga agg agc ctg ggt tcc ctg acc att gct gag 1252 Ser Leu Ala Arg Gly Arg Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu 75 80 85 90 ccg gcc atg atc gcc gag tgc aag acg cgc acc gag gtg ttc gag atc 1300 Pro Ala Met Ile Ala Glu Cys Lys Thr Arg Thr Glu Val Phe Glu Ile 95 100 105 tcc cgg cgc ctc ata gac cgc acc aac gcc aac ttc ctg gtg tgg ccg 1348 Ser Arg Arg Leu Ile Asp Arg Thr Asn Ala Asn Phe Leu Val Trp Pro 110 115 120 ccc tgt gtg gag gtg cag cgc tgc tcc ggc tgc tgc aac aac cgc aac 1396 Pro Cys Val Glu Val Gln Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn 125 130 135 gtg cag tgc cgc ccc acc cag gtg cag ctg cga cct gtc cag gtg aga 1444 Val Gln Cys Arg Pro Thr Gln Val Gln Leu Arg Pro Val Gln Val Arg 140 145 150 aag atc gag att gtg cgg aag aag cca atc ttt aag aag gcc acg gtg 1492 Lys Ile Glu Ile Val Arg Lys Lys Pro Ile Phe Lys Lys Ala Thr Val 155 160 165 170 acg ctg gaa gac cac ctg gca tgc aag tgt gag aca gtg gca gct gca 1540 Thr Leu Glu Asp His Leu Ala Cys Lys Cys Glu Thr Val Ala Ala Ala 175 180 185 cgg cct gtg acc cga agc ccg ggg ggt tcc cag gag cag cga gcc aaa 1588 Arg Pro Val Thr Arg Ser Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys 190 195 200 acg ccc caa act cgg gtg acc att cgg acg gtg cga gtc cgc cgg ccc 1636 Thr Pro Gln Thr Arg Val Thr Ile Arg Thr Val Arg Val Arg Arg Pro 205 210 215 ccc aag ggc aag cac cgg aaa ttc aag cac acg cat gac aag acg gca 1684 Pro Lys Gly Lys His Arg Lys Phe Lys His Thr His Asp Lys Thr Ala 220 225 230 ctg aag gag acc ctt gga gcc taggggcatc ggcaggagag tgtgtgggca 1735 Leu Lys Glu Thr Leu Gly Ala 235 240 gggttattta atatggtatt tgctgtattg cccccatggg gccttggagt agataatatt 1795 gtttccctcg tccgtctgtc tcgatgcctg attcggacgg ccaatggtgc ctcccccacc 1855 cctccacgtg tccgtccacc cttccatcag cgggtctcct cccagcggcc tccggctctt 1915 gcccagcagc tcaagaagaa aaagaaggac tgaactccat cgccatcttc ttcccttaac 1975 tccaagaact tgggataaga gtgtgagaga gactgatggg gtcgctcttt gggggaaacg 2035 ggttccttcc cctgcacctg gcctgggcca cacctgagcg ctgtggactg tcctgaggag 2095 ccctgaggac ctctcagcat agcctgcctg atccctgaac cc 2137 26 241 PRT Homo sapiens 26 Met Asn Arg Cys Trp Ala Leu Phe Leu Ser Leu Cys Cys Tyr Leu Arg 1 5 10 15 Leu Val Ser Ala Glu Gly Asp Pro Ile Pro Glu Glu Leu Tyr Glu Met 20 25 30 Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu 35 40 45 His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met 50 55 60 Thr Arg Ser His Ser Gly Gly Glu Leu Glu Ser Leu Ala Arg Gly Arg 65 70 75 80 Arg Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu 85 90 95 Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp 100 105 110 Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln 115 120 125 Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr 130 135 140 Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg 145 150 155 160 Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu 165 170 175 Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser 180 185 190 Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr Arg Val 195 200 205 Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg 210 215 220 Lys Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly 225 230 235 240 Ala 27 3007 DNA Homo sapiens CDS (492)..(1529) 27 gcccggagag ccgcatctat tggcagcttt gttattgatc agaaactgct cgccgccgac 60 ttggcttcca gtctggctgc gggcaaccct tgagttttcg cctctgtcct gtcccccgaa 120 ctgacaggtg ctcccagcaa cttgctgggg acttctcgcc gctcccccgc gtccccaccc 180 cctcattcct ccctcgcctt cacccccacc cccaccactt cgccacagct caggatttgt 240 ttaaaccttg ggaaactggt tcaggtccag gttttgcttt gatccttttc aaaaactgga 300 gacacagaag agggctctag gaaaaagttt tggatgggat tatgtggaaa ctaccctgcg 360 attctctgct gccagagcag gctcggcgct tccaccccag tgcagccttc ccctggcggt 420 ggtgaaagag actcgggagt cgctgcttcc aaagtgcccg ccgtgagtga gctctcaccc 480 cagtcagcca a atg agc ctc ttc ggg ctt ctc ctg ctg aca tct gcc ctg 530 Met Ser Leu Phe Gly Leu Leu Leu Leu Thr Ser Ala Leu 1 5 10 gcc ggc cag aga cag ggg act cag gcg gaa tcc aac ctg agt agt aaa 578 Ala Gly Gln Arg Gln Gly Thr Gln Ala Glu Ser Asn Leu Ser Ser Lys 15 20 25 ttc cag ttt tcc agc aac aag gaa cag aac gga gta caa gat cct cag 626 Phe Gln Phe Ser Ser Asn Lys Glu Gln Asn Gly Val Gln Asp Pro Gln 30 35 40 45 cat gag aga att att act gtg tct act aat gga agt att cac agc cca 674 His Glu Arg Ile Ile Thr Val Ser Thr Asn Gly Ser Ile His Ser Pro 50 55 60 agg ttt cct cat act tat cca aga aat acg gtc ttg gta tgg aga tta 722 Arg Phe Pro His Thr Tyr Pro Arg Asn Thr Val Leu Val Trp Arg Leu 65 70 75 gta gca gta gag gaa aat gta tgg ata caa ctt acg ttt gat gaa aga 770 Val Ala Val Glu Glu Asn Val Trp Ile Gln Leu Thr Phe Asp Glu Arg 80 85 90 ttt ggg ctt gaa gac cca gaa gat gac ata tgc aag tat gat ttt gta 818 Phe Gly Leu Glu Asp Pro Glu Asp Asp Ile Cys Lys Tyr Asp Phe Val 95 100 105 gaa gtt gag gaa ccc agt gat gga act ata tta ggg cgc tgg tgt ggt 866 Glu Val Glu Glu Pro Ser Asp Gly Thr Ile Leu Gly Arg Trp Cys Gly 110 115 120 125 tct ggt act gta cca gga aaa cag att tct aaa gga aat caa att agg 914 Ser Gly Thr Val Pro Gly Lys Gln Ile Ser Lys Gly Asn Gln Ile Arg 130 135 140 ata aga ttt gta tct gat gaa tat ttt cct tct gaa cca ggg ttc tgc 962 Ile Arg Phe Val Ser Asp Glu Tyr Phe Pro Ser Glu Pro Gly Phe Cys 145 150 155 atc cac tac aac att gtc atg cca caa ttc aca gaa gct gtg agt cct 1010 Ile His Tyr Asn Ile Val Met Pro Gln Phe Thr Glu Ala Val Ser Pro 160 165 170 tca gtg cta ccc cct tca gct ttg cca ctg gac ctg ctt aat aat gct 1058 Ser Val Leu Pro Pro Ser Ala Leu Pro Leu Asp Leu Leu Asn Asn Ala 175 180 185 ata act gcc ttt agt acc ttg gaa gac ctt att cga tat ctt gaa cca 1106 Ile Thr Ala Phe Ser Thr Leu Glu Asp Leu Ile Arg Tyr Leu Glu Pro 190 195 200 205 gag aga tgg cag ttg gac tta gaa gat cta tat agg cca act tgg caa 1154 Glu Arg Trp Gln Leu Asp Leu Glu Asp Leu Tyr Arg Pro Thr Trp Gln 210 215 220 ctt ctt ggc aag gct ttt gtt ttt gga aga aaa tcc aga gtg gtg gat 1202 Leu Leu Gly Lys Ala Phe Val Phe Gly Arg Lys Ser Arg Val Val Asp 225 230 235 ctg aac ctt cta aca gag gag gta aga tta tac agc tgc aca cct cgt 1250 Leu Asn Leu Leu Thr Glu Glu Val Arg Leu Tyr Ser Cys Thr Pro Arg 240 245 250 aac ttc tca gtg tcc ata agg gaa gaa cta aag aga acc gat acc att 1298 Asn Phe Ser Val Ser Ile Arg Glu Glu Leu Lys Arg Thr Asp Thr Ile 255 260 265 ttc tgg cca ggt tgt ctc ctg gtt aaa cgc tgt ggt ggg aac tgt gcc 1346 Phe Trp Pro Gly Cys Leu Leu Val Lys Arg Cys Gly Gly Asn Cys Ala 270 275 280 285 tgt tgt ctc cac aat tgc aat gaa tgt caa tgt gtc cca agc aaa gtt 1394 Cys Cys Leu His Asn Cys Asn Glu Cys Gln Cys Val Pro Ser Lys Val 290 295 300 act aaa aaa tac cac gag gtc ctt cag ttg aga cca aag acc ggt gtc 1442 Thr Lys Lys Tyr His Glu Val Leu Gln Leu Arg Pro Lys Thr Gly Val 305 310 315 agg gga ttg cac aaa tca ctc acc gac gtg gcc ctg gag cac cat gag 1490 Arg Gly Leu His Lys Ser Leu Thr Asp Val Ala Leu Glu His His Glu 320 325 330 gag tgt gac tgt gtg tgc aga ggg agc aca gga gga tag ccgcatcacc 1539 Glu Cys Asp Cys Val Cys Arg Gly Ser Thr Gly Gly 335 340 345 accagcagct cttgcccaga gctgtgcagt gcagtggctg attctattag agaacgtatg 1599 cgttatctcc atccttaatc tcagttgttt gcttcaagga cctttcatct tcaggattta 1659 cagtgcattc tgaaagagga gacatcaaac agaattagga gttgtgcaac agctcttttg 1719 agaggaggcc taaaggacag gagaaaaggt cttcaatcgt ggaaagaaaa ttaaatgttg 1779 tattaaatag atcaccagct agtttcagag ttaccatgta cgtattccac tagctgggtt 1839 ctgtatttca gttctttcga tacggcttag ggtaatgtca gtacaggaaa aaaactgtgc 1899 aagtgagcac ctgattccgt tgccttgctt aactctaaag ctccatgtcc tgggcctaaa 1959 atcgtataaa atctggattt tttttttttt ttttgctcat attcacatat gtaaaccaga 2019 acattctatg tactacaaac ctggttttta aaaaggaact atgttgctat gaattaaact 2079 tgtgtcgtgc tgataggaca gactggattt ttcatatttc ttattaaaat ttctgccatt 2139 tagaagaaga gaactacatt catggtttgg aagagataaa cctgaaaaga agagtggcct 2199 tatcttcact ttatcgataa gtcagtttat ttgtttcatt gtgtacattt ttatattctc 2259 cttttgacat tataactgtt ggcttttcta atcttgttaa atatatctat ttttaccaaa 2319 ggtatttaat attctttttt atgacaactt agatcaacta tttttagctt ggtaaatttt 2379 tctaaacaca attgttatag ccagaggaac aaagatgata taaaatattg ttgctctgac 2439 aaaaatacat gtatttcatt ctcgtatggt gctagagtta gattaatctg cattttaaaa 2499 aactgaattg gaatagaatt ggtaagttgc aaagactttt tgaaaataat taaattatca 2559 tatcttccat tcctgttatt ggagatgaaa ataaaaagca acttatgaaa gtagacattc 2619 agatccagcc attactaacc tattcctttt ttggggaaat ctgagcctag ctcagaaaaa 2679 cataaagcac cttgaaaaag acttggcagc ttcctgataa agcgtgctgt gctgtgcagt 2739 aggaacacat cctatttatt gtgatgttgt ggttttatta tcttaaactc tgttccatac 2799 acttgtataa atacatggat atttttatgt acagaagtat gtctcttaac cagttcactt 2859 attgtactct ggcaatttaa aagaaaatca gtaaaatatt ttgcttgtaa aatgcttaat 2919 atcgtgccta ggttatgtgg tgactatttg aatcaaaaat gtattgaatc atcaaataaa 2979 agaatgtggc tattttgggg agaaaatt 3007 28 345 PRT Homo sapiens 28 Met Ser Leu Phe Gly Leu Leu Leu Leu Thr Ser Ala Leu Ala Gly Gln 1 5 10 15 Arg Gln Gly Thr Gln Ala Glu Ser Asn Leu Ser Ser Lys Phe Gln Phe 20 25 30 Ser Ser Asn Lys Glu Gln Asn Gly Val Gln Asp Pro Gln His Glu Arg 35 40 45 Ile Ile Thr Val Ser Thr Asn Gly Ser Ile His Ser Pro Arg Phe Pro 50 55 60 His Thr Tyr Pro Arg Asn Thr Val Leu Val Trp Arg Leu Val Ala Val 65 70 75 80 Glu Glu Asn Val Trp Ile Gln Leu Thr Phe Asp Glu Arg Phe Gly Leu 85 90 95 Glu Asp Pro Glu Asp Asp Ile Cys Lys Tyr Asp Phe Val Glu Val Glu 100 105 110 Glu Pro Ser Asp Gly Thr Ile Leu Gly Arg Trp Cys Gly Ser Gly Thr 115 120 125 Val Pro Gly Lys Gln Ile Ser Lys Gly Asn Gln Ile Arg Ile Arg Phe 130 135 140 Val Ser Asp Glu Tyr Phe Pro Ser Glu Pro Gly Phe Cys Ile His Tyr 145 150 155 160 Asn Ile Val Met Pro Gln Phe Thr Glu Ala Val Ser Pro Ser Val Leu 165 170 175 Pro Pro Ser Ala Leu Pro Leu Asp Leu Leu Asn Asn Ala Ile Thr Ala 180 185 190 Phe Ser Thr Leu Glu Asp Leu Ile Arg Tyr Leu Glu Pro Glu Arg Trp 195 200 205 Gln Leu Asp Leu Glu Asp Leu Tyr Arg Pro Thr Trp Gln Leu Leu Gly 210 215 220 Lys Ala Phe Val Phe Gly Arg Lys Ser Arg Val Val Asp Leu Asn Leu 225 230 235 240 Leu Thr Glu Glu Val Arg Leu Tyr Ser Cys Thr Pro Arg Asn Phe Ser 245 250 255 Val Ser Ile Arg Glu Glu Leu Lys Arg Thr Asp Thr Ile Phe Trp Pro 260 265 270 Gly Cys Leu Leu Val Lys Arg Cys Gly Gly Asn Cys Ala Cys Cys Leu 275 280 285 His Asn Cys Asn Glu Cys Gln Cys Val Pro Ser Lys Val Thr Lys Lys 290 295 300 Tyr His Glu Val Leu Gln Leu Arg Pro Lys Thr Gly Val Arg Gly Leu 305 310 315 320 His Lys Ser Leu Thr Asp Val Ala Leu Glu His His Glu Glu Cys Asp 325 330 335 Cys Val Cys Arg Gly Ser Thr Gly Gly 340 345 29 399 DNA Orf virus CDS (1)..(399) 29 atg aag ttt ctc gtc ggc ata ctg gta gct gtg tgc ttg cac cag tat 48 Met Lys Phe Leu Val Gly Ile Leu Val Ala Val Cys Leu His Gln Tyr 1 5 10 15 ctg ctg aac gcg gac agc acg aaa aca tgg tcc gaa gtg ttt gaa aac 96 Leu Leu Asn Ala Asp Ser Thr Lys Thr Trp Ser Glu Val Phe Glu Asn 20 25 30 agc ggg tgc aag cca agg ccg atg gtc ttt cga gta cac gac gag cac 144 Ser Gly Cys Lys Pro Arg Pro Met Val Phe Arg Val His Asp Glu His 35 40 45 ccg gag cta act tct cag cgg ttc aac ccg ccg tgt gtc acg ttg atg 192 Pro Glu Leu Thr Ser Gln Arg Phe Asn Pro Pro Cys Val Thr Leu Met 50 55 60 cga tgc ggc ggg tgc tgc aac gac gag agc tta gaa tgc gtc ccc acg 240 Arg Cys Gly Gly Cys Cys Asn Asp Glu Ser Leu Glu Cys Val Pro Thr 65 70 75 80 gaa gag gca aac gta acg atg caa ctc atg gga gcg tcg gtc tcc ggt 288 Glu Glu Ala Asn Val Thr Met Gln Leu Met Gly Ala Ser Val Ser Gly 85 90 95 ggt aac ggg atg caa cat ctg agc ttc gta gag cat aag aaa tgc gat 336 Gly Asn Gly Met Gln His Leu Ser Phe Val Glu His Lys Lys Cys Asp 100 105 110 tgt aaa cca cca ctc acg acc acg cca ccg acg acc aca agg ccg ccc 384 Cys Lys Pro Pro Leu Thr Thr Thr Pro Pro Thr Thr Thr Arg Pro Pro 115 120 125 aga aga cgc cgc tag 399 Arg Arg Arg Arg 130 30 132 PRT Orf virus 30 Met Lys Phe Leu Val Gly Ile Leu Val Ala Val Cys Leu His Gln Tyr 1 5 10 15 Leu Leu Asn Ala Asp Ser Thr Lys Thr Trp Ser Glu Val Phe Glu Asn 20 25 30 Ser Gly Cys Lys Pro Arg Pro Met Val Phe Arg Val His Asp Glu His 35 40 45 Pro Glu Leu Thr Ser Gln Arg Phe Asn Pro Pro Cys Val Thr Leu Met 50 55 60 Arg Cys Gly Gly Cys Cys Asn Asp Glu Ser Leu Glu Cys Val Pro Thr 65 70 75 80 Glu Glu Ala Asn Val Thr Met Gln Leu Met Gly Ala Ser Val Ser Gly 85 90 95 Gly Asn Gly Met Gln His Leu Ser Phe Val Glu His Lys Lys Cys Asp 100 105 110 Cys Lys Pro Pro Leu Thr Thr Thr Pro Pro Thr Thr Thr Arg Pro Pro 115 120 125 Arg Arg Arg Arg 130

Claims

1-94. (canceled)

95. A method of stimulating stem cell recruitment, proliferation, or differentiation to stimulate myelopoiesis comprising,

identifying a mammalian subject in need of stem cell recruitment, proliferation, or differentiation to treat, prevent, or reduce myelopsuppression, and

administering to the mammalian subject a composition comprising a vascular endothelial growth factor B (VEGF-B) product, in an amount effective to stimulate myelopoiesis in the subject.

96. The method of claim 95, wherein the mammalian subject is human.

97. The method of claim 95, wherein the identifying comprises selecting a subject selected from the group consisting of:

(a) a subject undergoing antineoplastic chemotherapy;

(b) a bone marrow transplant subject; and

(c) a subject undergoing antineoplastic radiation therapy.

98. The method of claim 97, wherein the administering comprises administering the composition contemporaneously with, or after, administering at least one of the antineoplastic chemotherapy, the bone marrow transplant, and the antineoplastic radiation therapy.

99. The method of claim 95, wherein the identifying comprises:

measuring circulating white blood cells or bone-marrow derived stem cells in the subject to screen for myelosuppression.

100. The method of claim 99, wherein the measuring comprises measuring at least one of CD34+ stem cells and hematopoietic stem cells.

101. The method of any one of claims 95, wherein the method further comprises monitoring the number of circulating white blood cells or bone-marrow derived stem cells after administration of the composition.

102. The method of claim 101, wherein the monitoring comprises detection of at least one cell surface marker selected from the group consisting of VEGFR-1, VEGFR-2, and CD34.

103. The method of any one of claims 95, further comprising administering to said subject an agent selected from the group consisting of:

(a) granulocyte colony stimulating factor (G-CSF), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), interleukin-3 (IL-3), stem cell factor (SCF), vascular endothelial growth factor (VEGF), vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), platelet derived growth factor A (PDGF-A), platelet derived growth factor B (PDGF-B), platelet derived growth factor C (PDGF-C), platelet derived growth factor D (PDGF-D), and placental growth factor (PlGF);

(c) combinations thereof.

104. The method of claim 95, wherein the VEGF-B product comprises a VEGF-B polypeptide.

105. The method of claim 104, wherein the VEGF-B is glycosylated.

106. The method of claim 95, wherein the VEGF-B product comprises a polynucleotide that encodes a VEGF-B polypeptide.

107. The method of claim 106, wherein the VEGF-B product comprises a viral vector containing the polynucleotide.

108. The method of claim 107, wherein the vector comprises a replication-deficient adenoviral or adeno-associated viral vector.

109. The method of claim 104, wherein the VEGF-B polypeptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or a fragment thereof that binds VEGFR-1.

110. The method of claim 104, wherein the VEGF-B polypeptide is associated as a heterodimer with a VEGF polypeptide.

111. The method of claims 104, wherein the VEGF-B polypeptide binds VEGFR-1 and is encoded by a polynucleotide that hybridizes under stringent conditions with the complement of the polynucleotide in SEQ ID NO: 1 or 3.

112. The method of claim 95, wherein the VEGF-B product further comprises a pharmaceutically acceptable carrier.

113. A method of stimulating stem cell proliferation or differentiation, comprising,

obtaining a biological sample from a mammalian subject, wherein said sample comprises stem cells, and

contacting the stem cells with a composition comprising a vascular endothelial growth factor B (VEGF-B) product.

114. The method according to claim 113, further comprising a step of purifying and isolating the stem cells after obtaining the sample and before the contacting step.

115. The method according to claim 113, further comprising a step of purifying and isolating the stem cells after the contacting step.

116. The method according to claim 115, wherein the purified stem cells comprise stem cells selected from the group consisting of VEGFR-1+stem cells, CD34+stem cells, CD133+stem cells, and combinations of the same.

117. The method according to claims 113, wherein the contacting comprises culturing the stem cells in a culture containing the VEGF-B product.

118. The method according to any one of claims 113, further comprising a step of returning the stem cells to the mammalian subject.

119. The method according to any one of claims 113, further comprising a step of transplanting the cells into a different mammalian subject.

120. The method of claim 118, wherein the cells are seeded into a tissue, organ, or artificial matrices ex vivo, and said tissue, organ, or artificial matrix is attached, implanted, or transplanted into the mammalian subject.

121. The method of claim 119, wherein the cells are seeded into a tissue, organ, or artificial matrices ex vivo, and said tissue, organ, or artificial matrix is attached, implanted, or transplanted into the mammalian subject.

122. The method according to claim 113, wherein the mammalian subject is human.

123. The method according to claim 122, wherein the human subject needs antineoplastic chemotherapy, and wherein the biological sample is obtained prior to administering a dose of chemotherapy, and wherein the stem cells are returned to the human subject after the contacting and after the dose of chemotherapy.

124. The method according to any one of claims 113, wherein the VEGF-B product comprises a VEGF-B polypeptide.

125. The method of claim 124, wherein the VEGF-B is glycosylated.

126. The method of claim 113, wherein the VEGF-B product comprises a polynucleotide that encodes a VEGF-B polypeptide.

127. The method of claim 126, wherein the VEGF-B product comprises a viral vector containing the polynucleotide.

128. The method of claim 127, wherein the vector comprises a replication-deficient adenoviral or adeno-associated viral vector.

129. The method of claim 124, wherein the VEGF-B polypeptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or a fragment thereof that binds VEGFR-1.

130. The method of claim 124, wherein the VEGF-B polypeptide is associated as a heterodimer with a VEGF polypeptide.

131. The method of claims 124, wherein the VEGF-B polypeptide binds VEGFR-1 and is encoded by a polynucleotide that hybridizes under stringent conditions with the complement of the polynucleotide in SEQ ID NO: 1 or 3.

132. The method of claim 113, wherein the VEGF-B product further comprises a pharmaceutically acceptable carrier.

133. A method of stimulating stem cell recruitment, proliferation, or differentiation comprising,

identifying a mammalian subject in need of stem cell recruitment, proliferation, or differentiation to treat or prevent ischemia, and

administering to the subject a composition comprising a platelet derived growth factor (PDGF) product.

134. The method of claim 133, wherein the subject is human.

135. The method of claim 133, wherein the PDGF product comprises at least one member selected from the group consisting of PDGF-A, PDGF-B, PDGF-C, and PDGF-D products.

136. The method of claim 133, wherein the PDGF product binds PDGFR-α.

137. The method of claim 133, wherein the PDGF product comprises at least a PDGF-C product.

138. The method of claim 133, wherein the PDGF product comprises a PDGF polypeptide.

139. The method of claim 133, wherein the PDGF product comprises a polynucleotide that encodes a PDGF polypeptide.

140. The method of claim 139, wherein the PDGF product comprises a viral vector containing the polynucleotide.

141. The method of claim 140, wherein the vector comprises a replication-deficient adenoviral or adeno-associated viral vector.

142. The method of claim 138, wherein the PDGF polypeptide comprises a portion of the amino acid sequence set forth in SEQ ID NO: 7 or 9 that is effective to bind PDGFR-alpha or PDGFR-beta.

143. The method of claim 138, wherein the PDGF polypeptide binds PDGFR-alpha or PDGFR-beta and is encoded by a polynucleotide that hybridizes under stringent conditions with the complement of the polynucleotide in SEQ ID NO: 6 or 8.

144. The method claim 133, wherein the PDGF polypeptide comprises a member selected from the group consisting of a PDGF-A polypeptide, a PDGF-B polypeptide, a PDGF-C polypeptide, a PDGF-D polypeptide, combinations thereof, or fragments thereof that bind to at least one of PDGF receptors alpha and beta (PDGFR-alpha, PDGFR-beta).

145. The method of claims 133, wherein the PDGF polypeptide comprises a PDGF-C or PDGF-D polypeptide or a fragment thereof that binds to at least one of PDGF receptors alpha and beta (PDGFR-alpha, PDGFR-beta).

146. The method of claims 133, wherein the composition further comprises a pharmaceutically acceptable carrier.

147. The method of claims 133, further comprising administering to said subject an agent selected from the group consisting of:

(a) granulocyte colony stimulating factor (G-CSF), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), interleukin-3 (IL-3), stem cell factor (SCF), vascular endothelial growth factor (VEGF), vascular endothelial growth factor B (VEGF-B) vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), platelet derived growth factor A (PDGF-A), platelet derived growth factor B (PDGF-B), platelet derived growth factor C (PDGF-C), platelet derived growth factor D (PDGF-D), and placental growth factor (PlGF);

(c) combinations thereof.

148. A method of stimulating stem cell proliferation or differentiation, comprising,

contacting the stem cells with a composition comprising a platelet derived growth factor C (PDGF-C) product or platelet derived growth factor D (PDGF-D) product.

149. The method according to claim 148, further comprising contacting the cells with at least one additional PDGF product selected from the group consisting of a PDGF-A product, a PDGF-B product, a PDGF-C product and a PDGF-D product.

150. The method according to claim 148, further comprising a step of isolating the stem cells after obtaining the sample and before the contacting step.

151. The method according to claim 148, further comprising a step of purifying and isolating the stem cells after the contacting step.

152. The method according to claim 151, wherein the purified stem cells comprise cells that express PDGFR-alpha.

153. The method according to claim 151, wherein the purified stem cells comprise CD34+stem cells.

154. The method according to claim 148, wherein the contacting comprises culturing the stem cells in a culture containing the PDGF-C product or PDGF-D product.

155. The method according to claim 148, further comprising a step of returning the stem cells to the mammalian subject after the contacting step.

156. The method according to claim 148, further comprising a step of transplanting the cells into a different mammalian subject after the contacting step.

157. The method of claim 155, wherein the cells are seeded into a tissue, organ, or artificial matrix ex vivo, and said tissue, organ, or artificial matrix is attached, implanted, or transplanted into the mammalian subject.

158. The method of claim 156, wherein the cells are seeded into a tissue, organ, or artificial matrix ex vivo, and said tissue, organ, or artificial matrix is attached, implanted, or transplanted into the mammalian subject.

159. The method according to claims 148, wherein the mammalian subject is human.

160. The method according to claim 159, wherein the human subject needs antineoplastic chemotherapy, and wherein the biological sample is obtained prior to administering a dose of chemotherapy, and wherein the stem cells are returned to the human subject after the contacting and after the dose of chemotherapy.

161. The method of claim 148, wherein the PDGF-C product or PDGF-D product comprises a PDGF-C polypeptide or PDGF-D polypeptide.

162. The method of claim 148, wherein the product comprises a polynucleotide that encodes a PDGF-C polypeptide or a PDGF-D polypeptide.

163. The method of claim 162, wherein the product comprises a viral vector containing the polynucleotide.

164. The method of claim 163, wherein the vector comprises a replication-deficient adenoviral or adeno-associated viral vector.

165. The method of claims 161, wherein the polypeptide comprises a portion of the amino acid sequence set forth in SEQ ID NO: 7 or 9 that is effective to bind PDGFR-alpha or PDGFR-beta.

166. The method of claim 161, wherein the PDGF polypeptide binds PDGFR-alpha or PDGFR-beta and is encoded by a polynucleotide that hybridizes under stringent conditions with the complement of the polynucleotide in SEQ ID NO: 6 or 8.

167. The method of claim 148, wherein the composition further comprises a pharmaceutically acceptable carrier.

168. The method of claim 148, further comprising administering to said subject an agent selected from the group consisting of:

(a) granulocyte colony stimulating factor (G-CSF), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), interleukin-3 (IL-3), stem cell factor (SCF), vascular endothelial growth factor (VEGF), vascular endothelial growth factor B (VEGF-B), vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), platelet derived growth factor A (PDGF-A), platelet derived growth factor B (PDGF-B), platelet derived growth factor C (PDGF-C), platelet derived growth factor D (PDGF-D), and placental growth factor (PlGF);

(c) combinations thereof.

169. The method of claim 150, wherein the isolating comprises isolating AC133+/CD34+ cells from the biological sample.

170. The method according to claim 148, wherein the contacting comprises contacting the stem cells with the composition until stem cells differentiate into CD144+ cells.

171. The method according to claim 148, wherein the contacting comprises contacting the stem cells with the composition until stem cells differentiate into SMA+/CD144−/CD31−/CD34− cells.

172. The method according to claim 148, wherein the composition further comprises a VEGF-A product.

173. The method according to claim 148, wherein the contacting comprises culturing the stem cells in a culture containing the PDGF-C product.

174. The method according to claim 148, further comprising a step of returning the stem cells to the mammalian subject after the contacting step.

175. The method of claim 174, wherein the cells are seeded into a tissue, organ, or artificial matrix ex vivo, and said tissue, organ, or artificial matrix is attached or implanted into the mammalian subject.

176. The method according to claim 148, further comprising a step of transplanting the cells into a different mammalian subject after the contacting step.

177. The method of claim 176, wherein the cells are seeded into a tissue, organ, or artificial matrix ex vivo, and said tissue, organ, or artificial matrix is attached or transplanted into the different mammalian subject.

178. The method according to claim 148, wherein the mammalian subject is human.

179. The method according to claim 178, wherein the human subject has an ischemic condition.

180. The method of claim 161, wherein the polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 7 or 9 and binds to at least one receptor selected from PDGFR-alpha and PDGFR-beta.

181. The method of claim 161, wherein the polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 10 and binds to and/or activates at least one receptor selected from PDGFR-α/α and PDGFR-α/β.

182. The method of claim 180, wherein the PDGF-C polypeptide binds PDGFR-α and is encoded by a polynucleotide that hybridizes under stringent conditions with the complement of the polynucleotide in SEQ ID NO: 6.

183. The method of claim 161, wherein the polypeptide binds PDGFR-alpha or PDGFR-beta and is encoded by a polynucleotide that hybridizes under stringent conditions with the complement of the polynucleotide of SEQ ID NO: 6 or 8.

184. A method of stimulating stem cell proliferation or differentiation, comprising,

obtaining a biological sample from a mammalian subject, wherein said sample comprises stem cells;

contacting a first aliquot of the stem cells with a first composition comprising a first growth factor product selected from a VEGF-B product and PDGF-C product; and

contacting a second aliquot of the stem cells with a second composition comprising a second growth factor product independently selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, PDGF-A, PDGF-B, PDGF-C, and PlGF products,

wherein the first and second growth factor products are not the same.

185. The method of claim 184, wherein the first growth factor product is a PDGF-C product and the second growth factor product is a VEGF-A product.

186. A method of promoting differentiation of stem cells into both endothelial and smooth muscle cells, comprising:

obtaining a biological sample from a mammalian subject, wherein said sample comprises stem cells; and

contacting the cells with a composition comprising a platelet-derived growth factor-C (PDGF-C) product, in an amount and for a time sufficient to cause the cells to differentiate into both endothelial and smooth muscle cells.

187. The method according to claim 184, further comprising returning the cells to the mammalian subject after the contacting.

188. The method of claim 187, wherein the mammalian subject has an ischemic condition.

189. A method of ameliorating an ischemic condition comprising:

(a) diagnosing a mammalian subject with an ischemic condition;

(b) isolating a biological sample from the mammalian subject, wherein the biological sample comprises stem cells;

(c) contacting the cells with a composition comprising a platelet-derived growth factor-C (PDGF-C) product, in an amount and for a time sufficient to cause the cells to differentiate into both endothelial and smooth muscle cells; and

(d) returning the cells to the mammalian subject.

190. The method according to claim 189, wherein the returning comprises implanting or injecting the cells into or adjacent to ischemic tissue of the mammalian subject.